WO2018223185A1 - Methods of determining the likelihood of hepatitis b virus recrudescence - Google Patents

Methods of determining the likelihood of hepatitis b virus recrudescence Download PDF

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Publication number
WO2018223185A1
WO2018223185A1 PCT/AU2018/050560 AU2018050560W WO2018223185A1 WO 2018223185 A1 WO2018223185 A1 WO 2018223185A1 AU 2018050560 W AU2018050560 W AU 2018050560W WO 2018223185 A1 WO2018223185 A1 WO 2018223185A1
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percentage
motif
snvs
mutations
sites
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PCT/AU2018/050560
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French (fr)
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Robyn Lindley
Nathan HALL
Jared MAMROT
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Gmdx Co Pty Ltd
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Priority claimed from AU2017902147A external-priority patent/AU2017902147A0/en
Application filed by Gmdx Co Pty Ltd filed Critical Gmdx Co Pty Ltd
Publication of WO2018223185A1 publication Critical patent/WO2018223185A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates generally to methods of determining the likelihood of hepatitis B virus recrudescence in a subject.
  • HBV Hepatitis B virus
  • the virus particle contains an outer lipid envelope and an icosahedral nucleocapsid core composed of protein.
  • the nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity.
  • the outer envelope contains embedded proteins which are involved in viral binding of, and entry into, susceptible cells, predominantly hepatocytes of humans and other higher primates.
  • HBV infection is age-dependent and include asymptomatic infection, acute hepatitis B, chronic HBV infection, cirrhosis and hepatocellular carcinoma (HCC).
  • Acute hepatitis B occurs in approximately 1% of perinatal infections, 10% of early childhood infections (children aged 1 -5 years) and 30% of late infections (people aged >5 years) .
  • Fulminant hepatitis develops in 0.1 -0.6% of acute hepatitis cases, with mortality from fulminant hepatitis B being approximately 70%.
  • HBV infection is inversely related to the age of acquisition, occurring in approximately 80-90% of people infected perinatally, about 30% of children infected before the age of 6 years, and in ⁇ 5% of infections occurring in otherwise healthy adults.
  • the number of patients with chronic HBV infection is estimated to be 350-400 million worldwide, with more than one million deaths annually resulting from cirrhosis, liver failure and hepatocellular carcinoma. HBV infection is therefore a major global health problem.
  • Emtriva® fovir dipivoxil (e.g . Hepsera®), entecavir (e.g . Baraclude®), telbivudine (e.g. Tyzeka®), clevudine (e.g . Levovir®) and tenofovir disoproxil (e.g . Viread®) .
  • These agents significantly suppress the replication of HBV DNA to a very low level, leading to more favorable clinical outcomes, including a significant reduction in serious liver disease.
  • the present invention is predicated in part on the determination that the number, percentage, ratio and/or type of mutations that might be attributed to one or more endogenous dea minases in the genomic nucleic acid of a subject with HBV infection, compared to the genomic nucleic acid of a healthy subject, is predictive of the likelihood that the subject will experience HBV recrudescence.
  • the type and number of single nucleotide variations (SNVs) in a subject can be used to measure one or more genetic indicators of deaminase activity.
  • the methods disclosed herein provide a means for accurately predicting whether a subject will experience HBV recrudescence.
  • the methods are useful for determining the likelihood that a subject with chronic HBV infection can successfully terminate antiviral therapy without HBV recrudescence.
  • the methods can also be extended to therapeutic applications, whereby a course of therapy is prescribed based on the information provided by the predictive methods described herein.
  • the present invention provides method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject, the method comprising : analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs); measuring one or more genetic indicators of endogenous deaminase activity (e.g.
  • a method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject comprising : analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs) : measuring one or more genetic indicators of endogenous deaminase activity (e.g.
  • HBV recrudescence is likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity, or determining that HBV recrudescence is not likely when no genetic indicators of endogenous deaminase activity are outside predetermined normal range interval for those genetic indicators of endogenous deaminase activity.
  • the invention provides a method for determining the likelihood of HBV recrudescence in a subject, the method comprising : analysing the sequence of a nucleic acid molecule from a subject to detect SNVs; measuring one or more genetic indicators of endogenous deaminase activity (e.g.
  • the one or more genetic indicators of endogenous deaminase activity is selected from among the percentage of single nucleotide variations (SNVs) that are at a deaminase motif (optionally also specifying the type of mutation and/or codon context) ; the percentage of SNVs at MC-3 sites; the percentage of SNVs that include mutation of an adenine nucleotide; the percentage of SNVs that include mutation of a thymine nucleotide; the percentage of SNVs that include mutation of a cytosine nucleotide; the percentage of SNVs that include mutation of a guanine nucleotide; the ratio of the percentage of SNVs that include mutation of a cytosine nucleotide to the percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio); the ratio of the percentage of SNVs that include mutation of an adenine nucleotide to the percentage of SNV
  • the endogenous deaminase is selected from among activation-induced cytosine deaminase (AID), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 1 cytosine deaminase (AP0BEC1), AP0BEC3A, AP0BEC3B, AP0BEC3C, AP0BEC3D, AP0BEC3F, AP0BEC3G, AP0BEC3H and an adenosine deaminase acting on RNA (ADAR).
  • AID activation-induced cytosine deaminase
  • APOBEC catalytic polypeptide-like 1 cytosine deaminase
  • AP0BEC1 cytosine deaminase
  • AP0BEC1 catalytic polypeptide-like cytosine deaminase
  • AP0BEC1 catalytic polypeptid
  • the one or more genetic indicators of endogenous deaminase activity include the number or percentage of SNVs at an ADAR motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an AP0BEC3G motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an AP0BEC3B site (optionally also specifying the type of mutation and/or codon context); the number or percentage of the percentage of SNVs at MC-3 sites; and/or the number or percentage of SNVs resulting from mutation of an adenine nucleotide.
  • the methods comprise assessing one or more genetic indicators of endogenous deaminase activity
  • exemplary deaminase motifs include an AID motif selected from among motifs comprising the nucleic acid sequence WRC/GYW and WRCG/CGYW (wherein the underlined nucleotide is mutated) ; an ADAR motif comprising the nucleic acid sequence WA/TW; WAY/RTW; WTAW/WTAW; RAWA/TWTY; AAC/GTT; AA /TTT; ACA/TGT; CCA/TGG; CGA TCG; and CTA/JAG (wherein the underlined nucleotide is mutated); an APOBEC3G motif selected from among motifs comprising the nucleic acid sequence CC/CC, CG/CG, CG/CG, TCG/CGA, CCG/CGG and CGG
  • the one or more genetic indicators of deaminase activity that are assessed comprise a genetic indicator of ADAR activity, a genetic indicator of APOBEC3B activity and/or a genetic indicator of APOBEC3G activity.
  • the methods comprise assessing at least one or more genetic indicators of ADAR activity and at least one or more genetic indicators of APOBEC3B activity; at least one or more genetic indicators of ADAR activity and at least one or more genetic indicators of APOBEC3G activity; at least one or more genetic indicators of APOBEC3B activity and at least one or more genetic indicators of APOBEC3G activity; or at least one or more genetic indicators of ADAR activity, at least one or more genetic indicators of APOBEC3B activity and at least one or more genetic indicators of APOBEC3G activity.
  • a genetic indicator of ADAR activity may be selected from among the number or percentage of SNVs resulting from a mutation of A and the number or percentage of SNVs that are within an ADAR motif (e.g. the number or percentage of total SNVs that are within the motif WA/TW; number or percentage of total SNVs that are within the motif AAA/TTT; number or percentage of SNVs within the motif ACA/TGT that are A>C mutations; number or percentage of T>C mutations within the motif AAC/GTT that are at MC2 sites; number or percentage of SNVs within the motif CCA/TGG that are T>A; number or percentage of A>G mutations within the motif CGA/TCG that are at MCI sites; number or percentage of SNVs within the motif CTA/TAG that are T>A; number or percentage of all SNVs that are transitions at the WA/TW motif; number or percentage of SNVs within the WA/TW motif that are at MCI sites; number or percentage of SNVs within an ADAR motif
  • a genetic indicator of APOBEC3B activity can be the number or percentage of SNVs that are within an APOBEC3B motif (e.g. the number or percentage of mutations at motifs TCA/TGA, TC/GA and/or TCW/WGA; number or percentage of all SNVs that are transitions at the TCW/WGA motif; number or percentage of SNVs within the TCW/WGA motif that are at MCI sites; number or percentage of SNVs within the TCW/WGA motif that are at MC2 sites; number or percentage of SNVs within the TCW/WGA motif that are at MC3 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MCI sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MCI
  • a genetic indicator of APOBEC3G activity is the number or percentage of SNVs that are within an APOBEC3G motif (e.g. the number or percentage of mutations at motifs CC/GG, CG/CG, CG/CG, TCG/CGA, and/or CGG/CCGA; the number or percentage of C to T mutations at the CC motif; the number or percentage of C to T mutations at the CG motif; the number or percentage of G to A mutations at the CG motif; the number or percentage of mutations within the motif TCGA/TCGA that are G to T; number or percentage of all SNVs that are transitions at the CC/GG motif; number or percentage of SNVs within the CC/GG motif that are at MCI sites; number or percentage of SNVs within the CC/GG motif that are at MC2 sites; number or percentage of SNVs within the CC/GG motif that are at MC3 sites; number or percentage of C>T mutations within the CC/GG motif
  • the SNVs do not include known single nucleotide polymorphisms.
  • the nucleic acid molecule has been obtained from a blood or saliva sample from the subject.
  • the methods further comprise obtaining a biological sample (such as a blood or saliva sample) from the subject and extracting the nucleic acid molecule.
  • the subject has chronic HBV infection.
  • the subject is on antiviral therapy.
  • Antiviral therapy can include, for example, nucleoside/nucleotide therapy (e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy) or immunotherapy (e.g. interferon-alpha (IFN-a) therapy).
  • nucleoside/nucleotide therapy e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy
  • immunotherapy e.g. interferon-alpha (IFN-a) therapy.
  • the methods of the present invention may further comprise providing a recommendation to the subject to continue antiviral therapy if it is determined that HBV recrudescence is likely, or further comprise providing a recommendation to the subject to terminate antiviral therapy if it is determined that HBV recrudescence is not likely.
  • Antiviral therapy can include, for example, nucleoside/nucleotide therapy (e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy) or immunotherapy (e.g . interferon-alpha (IFN-a) therapy).
  • nucleoside/nucleotide therapy e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy
  • immunotherapy e.g . interferon-alpha (IFN-a) therapy.
  • the invention relates to a method for treating HBV infection in a subject, comprising : performing the method described above a nd herein to determine whether HBV recrudescence is likely, and exposing the subject to an antiviral therapy if it is determined that HBV recrudescence is likely.
  • the present invention is directed to a method for treating HBV infection in a subject, comprising : (a) sending a biological sample obtained from a subject to a laboratory to (i) conduct the method of described above and herein to determine whether HBV recrudescence is likely, and (ii) provide the results of the method, wherein the results comprise a determination of whether HBV recrudescence is likely or unlikely; (b) receiving the results from step (a); and (c) exposing the subject to an antiviral therapy if the results comprise a determination that HBV recrudescence is likely.
  • a genetic indicator means one genetic indicator or more than one genetic indicator.
  • biological sample refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a subject or patient.
  • the biological sample is selected from any part of a patient's body, including, but not limited to bodily fluids such as saliva or blood, tissue, cells, hair, skin and nails.
  • the term "codon context" with reference to a mutation refers to the nucleotide position within a codon at which the mutation occurs.
  • the nucleotide positions within a mutated codon are annotated MC-1, MC-2 and MC-3, and refer to the first, second and third nucleotide positions, respectively, when the sequence of the codon is read 5' to 3'.
  • the phrase "determining the codon context of a mutation” or similar phrase means determining at which nucleotide position within the mutated codon the mutation occurs, i.e. , MC-1, MC-2 or MC-3.
  • control subject may refer to a subject known to be affected with HBV (positive control), or to a subject known to be not affected or diagnosed with HBV (negative control). It should be noted that a control subject that is known to be healthy, i.e. , not having an HBV infection, may possibly suffer from another disease not tested/known . It is also understood that control subjects and healthy controls include data obtained and used as a standard, i.e., it can be used over and over again for multiple different subjects.
  • the data from the control sample could have been obtained in a different set of experiments, for example, it could be an average obtained from a number of healthy subjects and not actually obtained at the time the data for the subject was obtained.
  • the term "correlating" generally refers to determining a relationship between one type of data with another or with a state.
  • correlating deaminase activity with the likelihood that a subject will experience HBV recrudescence comprises assessing genetic indicators of deaminase activity in a subject and comparing the levels of these indicators to genetic indicators of deaminase activity in persons known to be free of HBV infection or to predetermined normal range intervals for genetic indicators of deaminase activity.
  • gene is meant a unit of inheritance that occupies a specific locus on a genome and comprises transcriptional and/or translational regulatory sequences a nd/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).
  • a “genetic indicator of deaminase activity” refers to a number, percentage, ratio and/or type of a single nucleotide variation (SNV) that may be reflective of the activity of one or more endogenous deaminases.
  • SNV single nucleotide variation
  • Exemplary genetic indicators of deaminase activity include, but are not limited to, the number or percentage of SNVs at a deaminase motif; the number or percentage of SNVs at MC-3 sites; the number or percentage of SNVs that include mutation of an adenine nucleotide; the number or percentage of SNVs that include mutation of a thymine nucleotide; the number or percentage of SNVs that include mutation of a cytosine nucleotide; the number or percentage of SNVs that include mutation of a guanine nucleotide; the ratio of the number or percentage of SNVs that include mutation of a cytosine nucleotide to the number or percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio); the ratio of the number or percentage of SNVs that include mutation of an adenine nucleotide to the number or percentage of SNVs that include mutation of a thymine nucleotide (
  • the term "likelihood" or grammatical variations is used as a measure of whether HBV recrudescence will occur, and of whether subjects with a genetic indicator of deaminase activity that is outside a predetermined normal range interval, or with a particular sample profile of genetic indicators, will experience HBV recrudescence based on a given mathematical model.
  • An increased likelihood for example may be relative or absolute and may be expressed qualitatively or quantitatively.
  • an increased likelihood or risk that a subject will experience HBV recrudescence may be expressed as determining whether any genetic indicators of deaminase activity are identified as being outside the normal range interval (as taught herein) and placing the test subject in an "increased likelihood or risk" category, based upon previous population studies.
  • the methods comprise comparing a score based on the number of genetic indicators of deaminase activity that are outside a predetermined normal range interval to a "threshold score".
  • the threshold score is one that provides an acceptable ability to predict the likelihood of HBV recrudescence in a subject, and can be determined by those skilled in the art using any acceptable means.
  • receiver operating characteristic (ROC) curves are calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population has a first condition or risk and a second population has a second condition or risk (called arbitrarily, for example, "controlled infection” and "HBV recrudescence", or "low risk” and "high risk”).
  • a distribution of number of number of genetic indicators of deaminase activity that are outside a predetermined normal range interval in subjects who experience disease progression and in subjects who do not experience disease progression may overlap. Under such conditions, a test does not absolutely distinguish between disease progression and disease stability or regression with 100% accuracy. A threshold is selected, above which the test is considered to be "positive” and below which the test is considered to be “negative.”
  • the area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, for example, Hanley et a/, Radiology 143 : 29-36 (1982)).
  • AUC area under the curve
  • ROC receiver operating characteristic
  • the true positive and false positive rates for the data are calculated.
  • the sensitivity is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases.
  • the specificity is determined by counting the number of controls below the value for that feature and then dividing by the total number of controls.
  • ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to produce a single value, and this single value can be plotted in a ROC curve. Additionally, any combination of multiple features (e.g. , one or more other epigenetic markers), in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test.
  • the ROC curve is the plot of the sensitivity of a test against the specificity of the test, where sensitivity is traditionally presented on the vertical axis and specificity is traditionally presented on the horizontal axis.
  • AUC ROC values are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one.
  • An AUC ROC value may be thought of as equivalent to the Mann - Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.
  • diagnosis refers to the process of determining the likelihood of HBV recrudescence.
  • An assessment of genetic indicators of deaminase activity in a nucleic acid present in a biological sample from a subject is used to determine the likelihood of HBV recrudescence in a subject.
  • disease progression refers to a worsening of a disease over time. Disease progression can be associated with or measured by an increase or worsening of one or more symptoms or markers associated with the disease (e.g. an increase in serum alanine aminotransferase levels) . In instances where the disease is an infection, disease progression can also be associated with or measured by an increase in viral, bacterial or fungal load (e.g. an increase in detectable serum HBV).
  • disease progression refers to a worsening of a disease over time. Disease progression can be associated with or measured by an increase or worsening of one or more symptoms or markers associated with the disease (e.g. an increase in serum alanine aminotransferase levels) . In instances where the disease is an infection, disease progression can also be associated with or measured by an increase in viral, bacterial or fungal load (e.g. an increase in detectable serum HBV).
  • a "healthy" subject is a subject that does not have HBV infection.
  • level with reference to a SNV or genetic indicator of deaminase activity refers to the number, percentage, amount or ratio of SNV or genetic indicator of deaminase activity.
  • a “mutation type” refers to the specific nucleotide substitution that comprises the mutation, and is selected from among C to T, C to A, C to G, G to T, G to A, G to C, A to T, A to C, A to G, T to A, T to C and T to G mutations.
  • a mutation type of C to T refers to a mutation in which the targeted or mutated nucleotide C is replaced with the substituting nucleotide T.
  • the "nucleic acid” as used herein designates DNA, cDNA, mRNA, RNA, rRNA or cRNA. The term typically refers to polynucleotides greater than 30 nucleotide residues in length.
  • a "predetermined normal range interval" refers to a range of values, with an upper and lower limit, for a genetic indicator of deaminase activity that are reflective of a healthy or non-disease state.
  • the predetermined normal range interval can be determined by assessing a genetic indicator of deaminase activity in two or more non-diseased or healthy subjects (e.g. subjects that do not have HBV infection and/or who do not have any known infection or disease).
  • a normal range interval for the genetic indicator is then calculated to set the upper and lower limits of what would be considered normal values for that indicator, e.g. values that would reflect a normal deaminase activity in healthy individuals.
  • the normal range interval is calculated by measuring the average plus or minus 2 standard deviations, whereby the lower limit of the range interval is the average minus 2 standard deviations and the upper limit of the range interval is the average plus 2 standard deviations.
  • less than or more than 2 standard deviations is used to set the upper and lower limits of the interval, such as 1, 1.5, 2.5, 3, 3.5 or more standard deviations.
  • the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves.
  • ROC receiver operating characteristic
  • the subjects used to determine the predetermined normal range interval can be of any age, sex or background, or may be of a particular age, sex, ethnic background or other subpopulation.
  • two or more predetermined normal range intervals can be calculated for the same genetic indicator of deaminase activity, whereby each range interval is specific for a particular subpopulation, e.g. a particular sex, age group, ethnic background and/or other subpopulation.
  • recrudescence refers to the reappearance of an HBV infection after it has been quiescent i.e. after a period of latency or relative inactivity. Recrudescence of HBV can be assessed by direct detection of HBV DNA, RNA and/or antigen, and/or by indirect detection of other markers, such as serum alanine aminotransferase (ALT) levels. Recrudescence encompasses both short- and long-term reappearance of infection, sometimes also referred to as reactivation and relapse.
  • ALT serum alanine aminotransferase
  • reactivation refers to a sudden increase in HBV replication in a subject, and typically is confirmed when there is detectable serum HBV DNA of less than 2000 IU/mL and/or serum ALT of 1.2-5 x upper limit of normal (ULN) .
  • relapse refers to a sustained or increased reactivation of HBV.
  • detectable serum HBV DNA of greater than 2000 IU/ml, and/or serum ALT of greater than 5 x ULN for 16 weeks or more, or serum ALT of greater than 10 x ULN for 8 weeks or more is indicative of HBV relapse.
  • a "reference profile of genetic indicators of deaminase activity” provides an analysis in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genetic indictors of deaminase activity are evaluated.
  • the reference profile is associated with a defined risk or likelihood of HBV recrudescence, such as "no risk/likelihood”, negligible risk/likelihood”, “low risk/likelihood”, “medium risk/likelihood” or "high risk/likelihood”. Accordingly, by virtue of comparison to the reference profile, the risk of a subject experiencing HBV recrudescence can be determined, i.e. by obtaining the profile of genetic indicators of deaminase activity for the subject (i.e. the sample profile) and comparing it to the reference profile.
  • single nucleotide variation refers to a variation occurring in the sequence of a nucleic acid molecule (e.g. a subject nucleic acid molecule) compared to another nucleic acid molecule (e.g. a reference nucleic acid molecule or sequence), wherein the variation is a difference in the identity of a single nucleotide (e.g. A, T, C or G).
  • somatic mutation refers to a mutation in the DNA of somatic cells (i.e., not germ cells), occurring after conception.
  • somatic mutagenesis therefore refers to the process by which somatic mutations occur.
  • subject refers to any animal subject, particularly a mammalian subject. By way of an illustrative example, suitable subjects are humans. In some embodiments, the subject presents with clinical signs of a condition as defined herein. As used herein, the term “clinical sign”, or simply “sign”, refers to objective evidence of a disease present in a subject. Symptoms and/or signs associated with diseases referred to herein and the evaluation of such signs are routine and known in the art. Examples of signs of disease vary depending upon the disease. Signs of a HBV infection may include detection of HBV DNA, HBV antigen and/or elevated serum ALT levels. Typically, whether a subject has a disease, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the disease.
  • targeted somatic mutagenesis and “TSM” refer to the process of somatic mutagenesis resulting from one or more mutagenic agents, wherein mutagenesis occurs at a targeted nucleotide within a motif, the targeted nucleotide is present at a particular position within a codon (e.g.
  • the targeted nucleotide is mutated to a particular substituting nucleotide (i.e., the mutation is of a particular mutation type, e.g., C to T, not C to A or C to G) .
  • the type of mutation e.g., C to T
  • the motif at which the mutation occurs e.g. , WRC
  • codon context of the mutation i.e., the position within the codon at which the mutation occurs (e.g., MC-1, MC-2 or MC-3).
  • "Targeted somatic mutagen” therefore refers to mutation resulting from TSM.
  • the terms are also used herein to denote inhibiting the replication of HBV and/or alleviating the effects of HBV.
  • Those in need of treatment include those diagnosed with HBV, those suspected of having HBV or those at risk of getting HBV.
  • the subject to be treated herein may have been diagnosed as having the HBV or may be at risk of contracting HBV.
  • treatment refers to the eradication, removal, modification, or control of HBV infection that results from the administration of one or more therapeutic agents according to the methods of the invention.
  • treatment refers to the act of treating.
  • ADAR adenosine deaminases acting on RNA
  • AID activation-induced cytosine deaminase
  • APOBEC apolipoprotein B mRNA-editing enzyme, catalytic
  • APOBEC polypeptide-like cytosine deaminases
  • NTS non-transcribed strand
  • SHM somatic hypermutation
  • SNV single nucleotide variation
  • TS transcribed strand
  • TSM targeted somatic mutation
  • the present invention is predicated in part on the unexpected finding that the number, percentage, ratio and/or type of mutations that may be reflective of the activity of one or more endogenous deaminases in the genomic nucleic acid of a subject with HBV infection, compared to the genomic nucleic acid of a healthy subject, is predictive of the likelihood of HBV recrudescence in that subject.
  • the present invention encompasses methods for identifying these changes in endogenous deaminase activity as well as methods for determining the likelihood of HBV recrudescence in a subject by detecting these changes.
  • Endogenous deaminases are known to be involved in somatic mutagenesis, including somatic hypermutation and class switch recombination of immunoglobulin genes in B cells.
  • endogenous deaminases are key factors in RNA editing and innate immunity.
  • a number of deaminases have been shown to be involved in editing of viral RNA and DNA as a means to inhibit or reduce viral replication, and have also been implicated in activating other factors of innate immune response to combat viral infection, including (see e.g. Samuel (2012) Curr Top Microbiol Immunol. 353; Vieira and Soares (2013) BioMed Research International, 683095 ; He et al. (2015) Mol Med Rep. 12(5) : 6405-6414).
  • deaminases appear to have a proviral function (see e.g. Samuel (2011) Virology 411 : 180-193).
  • Endogenous deaminases include, for example, adenosine deaminases such as adenosine deaminases acting on RNA (ADAR), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytosine deaminases, and activation- induced cytosine deaminase (AID), and error-prone DNA polymerases such as DNA polymerase eta.
  • adenosine deaminases such as adenosine deaminases acting on RNA (ADAR), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytosine deaminases, and activation- induced cytosine deaminase (AID), and error-prone DNA polymerases such as DNA polymerase eta.
  • ADAR adenosine deaminases acting on RNA
  • TSM targeted somatic mutation
  • TSM refers to the process of somatic mutagenesis resulting from one or more mutagenic agents, such as a deaminase, wherein mutagenesis occurs at a targeted nucleotide within a motif, the targeted nucleotide is present at a particular position within a codon (e.g., the first, second or third position of the mutated codon reading from 5' to 3', annotated MC-1, MC-2 and MC-3, respectively), and the targeted nucleotide is mutated to a particular substituting nucleotide (i.e. , the mutation is of a particular mutation type, e.g. , C to T, not C to A or C to G).
  • mutagenic agents such as a deaminase
  • a determination that TSM is occurring requires analysis of the type of mutation (e.g., C to T), the motif at which the mutation occurs (e.g., WRC) and codon context of the mutation, i.e., the position within the codon at which the mutation occurs (e.g. , MC-1, MC-2 or MC-3).
  • the type of mutation e.g., C to T
  • the motif at which the mutation occurs e.g., WRC
  • codon context of the mutation i.e., the position within the codon at which the mutation occurs (e.g. , MC-1, MC-2 or MC-3).
  • Activation-induced cytosine deaminase is an important enzyme in adaptive immunity, involved in somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes in B cells.
  • SHM somatic hypermutation
  • AID triggers SHM by deaminating cytosines to uracils (C to U) to diversify the immunoglobulin variable region genes (VDJ) and create new antigen-binding sites. If unrepaired, the deamination of C to U by AID gives rise to C to T somatic mutations in DNA.
  • AID has been shown to be involved in the innate antiviral immune response, including editing the DNA and RNA of various viruses such as HBV (see e.g . He et al. (2015) Mol Med Rep. 12(5) : 6405-6414, Liang et al. Proc Natl Acad Sci U S A. 110(6) : 2246-51).
  • APOBEC cytosine deaminases that are known to be involved in innate immunity and RNA editing (Smith et al. (2012) Semin. Cell. Dev. Biol. 23: 258-268). In humans, at least AP0BEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H are involved in providing innate immunity and/or cellular mRNA editing. These APOBEC deaminases are also involved in the innate immune response, editing the DNA and RNA of various viruses (including HBV) and disrupting viral replication (see e.g. Turelli et al.
  • Double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes, are responsible for the type of RNA editing that is most prevalent in higher eukaryotes, i.e. conversion of adenosine residues into inosine (A-to-I editing).
  • ADAR is an enzyme that is encoded by the ADAR gene in humans.
  • the ADAR1 enzyme destabilizes dsRNA through conversion of adenosine to inosine.
  • the ADAR1 enzyme modifies cellular and viral RNA, including coding and noncoding RNAs.
  • ADAR1 is an RNA editing enzyme, required for hematopoiesis.
  • ADAR1 +/" chimeric embryos die before embryonic day 14 with defects in the hematopoietic system. Regulated levels of ADAR1 expression are critical for embryonic erythropoiesis in the liver. Mutations in the ADAR gene have been associated with dyschromatosis symmetrica hereditaria. Alternate transcriptional splice variants, encoding different isoforms, have been characterized.
  • ADARs Like other deaminases, ADARs have been implicated in the innate immune response to viruses. However, their precise role is in many instances not well defined, and in some examples, there appears to be both and antiviral and proviral role for ADARs (see e.g. Samuel (2011) Virology 411(2) : 180-93).
  • any one or more deaminases can be assessed as described herein by detecting mutations that are reflective of deaminase activity, such as ADAR, APOBEC3B and/or APOBEC3G activity.
  • single nucleotide variations SNVs
  • the number or percentage of these SNVs, and/or the relationship (e.g. the ratio) between these SNVs and other nucleotides in the nucleic acid molecule, may be indicative of deaminase activity. Accordingly, based on the type and/or number of the SNVs, genetic indicators of deaminase activity can be identified.
  • Genetic indicators of deaminase activity for use in the methods of the present invention include those described above and below. Exemplary of such indicators are, for example, the number or percentage of SNVs at a deaminase motif, optionally specified by mutation type and/or MC site (e.g.
  • the methods of the present invention comprise detecting SNVs at the targeted nucleotide in a deaminase motif.
  • the total number and/or percentage of total SNVs at a given motif, or at multiple motifs of a particular deaminase, is then calculated .
  • the motif may be any one or more motifs targeted by any one or more endogenous deaminases, including AID, ADAR, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and/or APOBEC3H .
  • the motifs may be on the non-transcribed and/or transcribed strand of the nucleic acid.
  • motifs are on the non-transcribed strand (NTS) of the nucleic acid.
  • Exemplary motifs utilized by AP0BEC3G include the motif comprising the nucleic acid sequence CC/GG; the motif comprising the nucleic acid sequence CG/CG; and the motif comprising the nucleic acid sequence CG/CG (wherein the underlined nucleotide is mutated).
  • Other motifs that can be targeted by AP0BEC3G include the motif comprising the nucleic acid sequence TCG/CGA; the motif comprising the nucleic acid sequence CCG; and the motif comprising the nucleic acid sequence CGG/CCG.
  • An exemplary AP0BEC3B motif comprises the nucleic acid sequence
  • a non-limiting example of a motif targeted by AP0BEC3H is one comprising a nucleic acid sequence GA/TC (wherein the underlined nucleotide is mutated).
  • Exemplary AP0BEC1 motifs include those comprising the nucleic acid sequence TG/CA or GG/CC (wherein the underlined nucleotide is mutated).
  • Exemplary of the motifs targeted by ADAR is the motif comprising the nucleic acid sequence WA/TW (wherein the underlined nucleotide is mutated).
  • Other exemplary motifs include the motif comprising the nucleic acid sequence WAY/RTW; the motif comprising the nucleic acid sequence RAWA/TWTY; the motif comprising the nucleic acid sequence WTAW/WTAW; the motif comprising the nucleic acid sequence SARA/TYTS; the motif comprising the nucleic acid sequence AAC/GTT; the motif comprising the nucleic acid sequence AAA/TTT; the motif comprising the nucleic acid sequence ACA TGT; the motif comprising the nucleic acid sequence CCA/TGG; the motif comprising the nucleic acid sequence CGA/TCG; and the motif comprising the nucleic acid sequence CTA/TAG.
  • WRC/GYW WRC/GYW
  • ADAR motif e.g. WA/TW
  • APOBEC3G e.g. CC/GG
  • APOBEC3B motif e.g. TCA/TGA
  • the methods of the present disclosure involve the detection of any mutation at the targeted nucleotide, e.g. an A, C or T off a G. Moreover, the methods typically involve the detection of a mutation when the targeted nucleotide is at any position within the codon (i.e. at MC-1, MC-2 or MC-3). Thus, a SNV at a deaminase motif is found to be present when there is any mutation at the targeted nucleotide within the motif and when the targeted nucleotide is at any position within the codon (as described in Examples 1-5, below).
  • detection of a SNV at a deaminase motif involves a further assessment of the type of a mutation and/or the codon context of a mutation before it is determined whether a SNV is attributable to deaminase activity.
  • a SNV at a deaminase motif is detected when there is a C to T mutation at the WRC AID motif; a G to A mutation at the GYW AID motif; an A to G mutation at the WA AID motif; an A to T mutation at the WA ADAR motif; a C to T mutation at the CC AP0BEC3G motif; a C to T mutation at the CG AP0BEC3G motif; a G to A mutation at the CG AP0BEC3G motif; a G to A mutation at the GA AP0BEC3H motif; a G to A mutation at the TG AP0BEC1 motif; or a G to T mutation at the GG AP0BEC1 motif; C to T mutations within the AID motif WRC/GYW that are at MCI sites; G to A mutations within the AID motif WRC/GYW that are at MC2 sites; C to T mutations within the motif ACA/TGT that are at
  • the methods may involve detection of targeted somatic mutation (i.e. including an assessment of both the type of mutation and the codon context of the mutation) to determine whether a SNV is attributable to a deaminase (as described in, for example, WO2014/066955 and WO2017/031551).
  • the methods of the present disclosure in some instances comprise detecting SNVs at the third position in the mutated codon, i.e. the MC-3 site.
  • the deaminases have a preference for targeting nucleotides at a particular position within the mutated codon .
  • the number and/or percentage of SNVs at a MC-3 site can be a genetic indicator of deaminase activity.
  • a detected aberration in this percentage is indicative of "off target" SHM, which can be associated with progressing oncogenesis.
  • A, T, C or G can also be used as a genetic indicator of deaminase activity given that particular deaminases can have a preference for targeting a particular nucleotide in a nucleic acid molecule.
  • adenosines are often the target of ADAR, while cytosines are often the target of AID.
  • the methods of the present disclosure can include determining the percentage of SNVs resulting from a mutation of an adenine nucleotide (i.e.
  • Evidence of strand bias of mutations can also be an indicator of deaminase activity.
  • Strand bias can be assessed by calculating various ratios of mutations targeting a particular nucleotide (described above). For example, the ratio of the percentage of SNVs that include mutation of a cytosine nucleotide to the percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio) can be determined. In another example, the ratio of the percentage of SNVs that include mutation of an adenine nucleotide to the percentage of SNVs that include mutation of a thymine nucleotide (A:T ratio) is determined.
  • the ratio of the percentage of SNVs that include mutation of an adenine or a thymine nucleotide to the percentage of SNVs that include mutation of a cytosine or a guanine nucleotide is determined.
  • mutations associated with ADAR activity can be particular useful in determining the likelihood of HBV recrudescence.
  • Non-limiting examples of genetic indicators of ADAR activity include the number or percentage of SNVs resulting from a mutation of A (i.e. A>T, A>G and A>C); and the number or percentage of SNVs that are within an ADAR motif such as one described above (e.g. the motif WA), optionally specifying the mutation type and/or the MC site, e.g.
  • Mutations associated with APOBEC activity, and in particular APOBEC3B or APOBEC3G activity, can also be particularly useful in determining the likelihood of HBV recrudescence.
  • Exemplary genetic indicators of APOBEC3B activity include the number or percentage of mutations that are within an APOBEC3B motif such as one described above, optionally specifying the mutation type and/or the MC site, e.g. the number or percentage of mutations at motifs TCA/TGA, TC/GA and/or TCW/WGA; number or percentage of all SNVs that are transitions at the TCW/WGA motif; number or percentage of SNVs within the TCW/WGA motif that are at MCI sites; number or percentage of SNVs within the TCW/WGA motif that are at MC2 sites; number or percentage of SNVs within the TCW/WGA motif that are at MC3 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MCI sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC3 sites; number or
  • Exemplary genetic indicators of APOBEC3G activity include the number or percentage of mutations that are within an APOBEC3G motif such as one described above, optionally specifying the mutation type and/or the MC site, e.g. the number or percentage of mutations at motifs CC/GG, CG/CG, CG/CG, TCG/CGA, and/or CGG/CCGA; the number or percentage of C to T mutations at the CC motif; the number or percentage of C to T mutations at the CG motif; the number or percentage of G to A mutations at the CG motif; the number or percentage of mutations within the motif TCGA/TCGA that are G to T; number or percentage of all SNVs that are transitions at the CC/GG motif; number or percentage of SNVs within the CC/GG motif that are at MCI sites; number or percentage of SNVs within the CC/GG motif that are at MC2 sites; number or percentage of SNVs within the CC/GG motif that are at MC3 sites
  • the nucleic acid molecule analysed using the methods of the present invention can be any nucleic acid molecule, although is generally DNA (including cDNA).
  • the nucleic acid is mammalian nucleic acid, such as human nucleic acid, and is from a subject previously diagnosed with HBV and undergoing antiviral therapy.
  • the nucleic acid can be obtained from any biological sample.
  • the biological sample may comprise a bodily fluid, tissue or cells.
  • the biological sample is a bodily fluid, such as saliva or blood.
  • the biological sample is a biopsy.
  • a biological sample comprising tissue or cells may from any part of the body and may comprise any type of cells or tissue, such as, for example, cells from the liver.
  • the nucleic acid molecule can contain a part or all of one gene, or a part or all of two or more genes, and it is the sequence of this gene or genes that is analysed according to the methods of the invention. Most typically, the nucleic acid molecule comprises the whole genome or whole exome, and it is the sequence of the whole genome or whole exome that is analysed in the methods of the invention.
  • the sequence of the nucleic acid molecule may have been predetermined.
  • the sequence may be stored in a database or other storage medium, and it is this sequence that is analysed according to the methods of the invention.
  • the sequence of the nucleic acid molecule must be first determined prior to employment of the methods of the invention.
  • the nucleic acid molecule must also be first isolated from the biological sample.
  • the biological sample from which the nucleic acid is obtained is a saliva sample or a blood sample.
  • a saliva sample or a blood sample there is strong concordance between the data generated from an analysis of nucleic acid obtained from saliva and the data generated from an analysis of nucleic acid obtained from blood of the same subject.
  • nucleic acid and/or sequenci ng the nucleic acid are well known in the art, and any such method can be utilized for the methods described herein.
  • the methods include amplification of the isolated nucleic acid prior to sequencing, and suitable nucleic acid amplification techniques are well known to a person of ordinary skill in the art.
  • Nucleic acid sequencing techniques are well known in the art and can be applied to single or multiple genes, or whole exomes or genomes. These techniques include, for example, capillary sequencing methods that rely upon 'Sanger sequencing' (Sanger et al.
  • next generation sequencing techniques that facilitate the sequencing of thousands to millions of molecules at once.
  • methods include, but are not limited to, pyrosequencing, which makes use of luciferase to read out signals as individual nucleotides are added to DNA templates; "sequencing by synthesis” technology (Illumina), which uses reversible dye- terminator techniques that add a single nucleotide to the DNA template in each cycle; and SOLiDTM sequencing (Sequencing by Oligonucleotide Ligation and Detection; Life Technologies), which sequences by preferential ligation of fixed-length oligonucleotides.
  • next generation sequencing techniques are particularly useful for sequencing whole exomes and genomes.
  • SNVs are then identified. SNVs may be identified by comparing the sequence to a reference sequence.
  • the reference sequence may be the sequence of a nucleic acid molecule from a database, such as reference genome.
  • the reference sequence is a reference genome, such as GRCh38 (hg38), GRCh37 (hg l9), NCBI Build 36.1 (hg l8), NCBI Build 35 (hg l7) and NCBI Build 34 (hg l6) .
  • SNPs single nucleotide polymorphisms
  • only those SNVs that are within a coding region of an ENSEMBL gene are selected for further analysis.
  • the codon containing the mutation and the position of the mutation within the codon (MC-1, MC-2 or MC-3) may be identified.
  • Nucleotides in the flanking 5' and 3' codons are also identified so as to identify the motifs.
  • the sequence of the non-transcribed strand (equivalent to the cDNA sequence) of the nucleic acid molecules is analysed .
  • the sequence of the transcribed strand is analysed.
  • kits comprising reagents to facilitate that isolation and/or sequencing are envisioned.
  • reagents can include, for example, primers for amplification of DNA, polymerase, dNTPs (including labelled dNTPs), positive and negative controls, and buffers and solutions.
  • kits will also generally comprise, in suitable means, distinct containers for each individual reagent.
  • the kit can also feature various devices, and/or printed instructions for using the kit.
  • the methods described generally herein a re performed, at least in part, by a processing system, such as a suitably programmed computer system.
  • a processing system such as a suitably programmed computer system.
  • a stand-alone computer with the microprocessor executing applications software allowing the above-described methods to be performed, may be used.
  • the methods can be performed, at least in part, by one or more processing systems operating as part of a distributed architecture.
  • a processing system can be used to identify mutation types, the codon context of a mutation and/or motifs within one or more nucleic acid sequences.
  • commands inputted to the processing system by a user assist the processing system in making these determinations.
  • a processing system includes at least one microprocessor, a memory, an input/output device, such as a keyboard and/or display, and an external interface, interconnected via a bus.
  • the external interface can be utilised for connecting the processing system to peripheral devices, such as a communications network, database, or storage devices.
  • the microprocessor can execute instructions in the form of applications software stored in the memory to allow the methods of the present invention to be performed, as well as to perform any other required processes, such as communicating with the computer systems.
  • the applications software may include one or more software modules, and may be executed in a suitable execution environment, such as an operating system environment, or the like.
  • the likelihood of HBV recrudescence in a subject can be determined.
  • Such a determination can facilitate the prescribing of a treatment regimen for a subject in whom HBV is likely to recrudesce.
  • the determination can assist with deciding whether antiviral treatment should begin, or whether current antiviral therapy should continue, be adjusted (e.g. be increased or reduced or altered in quality) or be stopped.
  • the methods can be used to obtain a profile of genetic indicators of deaminase activity for a subject, i.e. a sample profile, which can then be compared to a reference profile of genetic indicators of deaminase activity.
  • Profiles of the present disclosure reflect an evaluation of at least any 1 or more, typically 2 or more, genetic indicators of deaminase activity associated with HBV recrudescence as described above.
  • Reference profiles correlate with a risk or likelihood of HBV recrudescence and are typically predetermined, although can also be determined at the time of or after determining a sample profile.
  • Reference profiles are determined based on data obtained in the evaluation of genetic indicators of deaminase activity in individuals that have a known risk or likelihood of HBV recrudescence.
  • the reference profiles can be based on the data obtained in the evaluation of genetic indicators of deaminase activity over time in individuals that have chronic HBV, were on antiviral therapy and then stopped antiviral therapy, and relapsed .
  • the reference profile can correlate with, for example, a high risk of HBV recrudescence.
  • the reference profile is based on the data obtained in the evaluation of genetic indicators of deaminase activity over time in individuals that have chronic HBV, were on antiviral therapy and then stopped antiviral therapy, and recovered .
  • the reference profile correlates to, for example, no risk or a negligible or low risk of HBV recrudescence.
  • the individuals used to generate the reference profile may be age, gender and/or ethnicity matched or not.
  • a profile of genetic indicators of deaminase activity comprises at least one measurement.
  • the profile represents an assessment of at least 2 indicators (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more).
  • the indicators can be associated with the same or different deaminases.
  • reference profiles encompass classification models, such as those formed using machine leaning techniques.
  • Classification models can be formed using any suitable statistical classification or learning method that attempts to segregate bodies of data into classes based on objective parameters present in the data.
  • Classification methods may be either supervised or unsupervised . Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition : A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, the teachings of which are incorporated by reference.
  • Non-limiting examples of techniques that can be used to produce classification models include deep learning techniques such as Deep Boltzmann Machine, Deep Belief Networks, Convolutional Neural Networks, Stacked Auto Encoders; ensemble techniques such as Random Forest, Gradient Boosting Machines, Boosting, Bootstrapped Aggregation, AdaBoost, Stacked Generalization, Gradient Boosted Regression Trees; neural network techniques such as Radial Basis Function Network, Perceptron, Back- Propagation, Hopfield Network; regularization methods such as Ridge Regression, Least Absolute Shrinkage and Selection Operator, Elastic Net, Least Angle Regression; regression methods such as Linear Regression, Ordinary Least Squares Regression, Multiple Regression, Probit Regression, Stepwise Regression, Multivariate Adaptive Regression Splines, Locally Estimated Scatterplot Smoothing, Logistic Regression, Support Vector Machines, Poisson Regression, Negative Binomial Regression, Multinomial Logistic Regression; Bayesian techniques such as Naive Bayes, Average One
  • Data from individuals with chronic HBV who were on antiviral therapy and then stopped antiviral therapy, and either recovered or relapsed can be used to train a classification model .
  • Such data is typically referred to as a training data set.
  • the classification model can recognize patterns in data generated using unknown samples, e.g. the data from patients with chronic HBV used to generate the sample profiles.
  • the sample profile can then be applied to the classification model to classify the sample profile into classes, e.g. likely to experience HBV recrudescence or unlikely to experience HBV recrudescence.
  • HBV recrudescence will be determined to be likely when 1, 2, 3, 4, 5 or more genetic indicators of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity.
  • HBV recrudescence will be determined to be likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity.
  • HBV recrudescence will be determined to be unlikely when one or more of these genetic indicators of endogenous deaminase activity are within the respective predetermined range intervals.
  • HBV recrudescence will be determined to be unlikely when none of the genetic indicators of endogenous deaminase activity are within the respective predetermined range intervals (i.e. no genetic indicators of endogenous deaminase activity are outside a predetermined normal range interval for genetic indicators of endogenous deaminase).
  • a score is attributed to each genetic indicator of endogenous deaminase activity that is outside a predetermined range interval, and the total score is then calculated by adding all of the scores. HBV recrudescence will then be determined to be likely when the score is equal to or above a threshold score.
  • the score for each genetic indicator of endogenous deaminase activity may be the same or may be different (e.g. may be "weighted" such that one genetic indicator of endogenous deaminase activity that is outside a predetermined range interval might be given a score that is more than another genetic indicator of endogenous deaminase activity that is outside a predetermined range interval).
  • each genetic indicator of endogenous deaminase activity that is outside a predetermined range interval is given a score of 1.
  • the threshold score is 1 such that any score of 1 and above results in a determination that HBV recrudescence is likely.
  • the predetermined normal range interval for a genetic indicator of endogenous deaminase activity can be determined by assessing a genetic indicator of deaminase activity in two or more non-diseased or healthy subjects (e.g. subjects that do not have HBV infection and/or who do not have any other known infection or disease). A normal range interval for the genetic indicator is then calculated to set the upper and lower limits of what would be considered normal values for that indicator, e.g. values that would reflect a normal deaminase activity in healthy individuals.
  • the normal range interval is calculated by measuring the average plus or minus 2 standard deviations, whereby the lower limit of the range interval is the average minus 2 standard deviations and the upper limit of the range interval is the average plus 2 standard deviations. In other examples, less than or more than 2 standard deviations are used to set the upper and lower limits of the interval, such as 1, 1.5, 2.5, 3, 3.5 or more standard deviations.
  • the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves. In still further examples, the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves.
  • the subjects used to determine the predetermined normal range interval can be of any age, sex or background, or may be of a particular age, sex, ethnic background or other subpopulation.
  • two or more predetermined normal range intervals can be calculated for the same genetic indicator of deaminase activity, whereby each range interval is specific for a particular subpopulation, e.g. a particular sex, age group, ethnic background and/or other subpopulation.
  • the subject has chronic HBV infection, and the methods of the present disclosure are performed to determine the likelihood of HBV recrudescence in these subjects.
  • the subject has chronic HBV and is on antiviral therapy, and the methods of the present disclosure are performed to determine the likelihood of HBV recrudescence in these subjects if they terminate antiviral therapy.
  • the subject does not have chronic HBV infection.
  • the subject may have experienced or may be experiencing an acute HBV infection, and may appear to be resolving or to have resolved that infection (with or without antiviral therapy). The methods of the present disclosure can therefore be performed to determine the likelihood of HBV recrudescence in these subjects.
  • an analysis of genetic indicators of deaminase activity as described herein can also be used to assist in determining whether or not antiviral therapy is working, and/or in monitoring disease progression .
  • nucleic acid obtained from biological samples from a subject over time e.g. over time during therapy; before therapy has commenced and/or after therapy has commenced
  • the methods of the present invention also extend to therapeutic or preventative protocols.
  • existing antiviral protocols may be amended to reduce the antiviral therapy (e.g. reduce dosage), or to remove a subject from a therapy completely.
  • protocols designed to reduce that likelihood may be designed and applied to a subject. For example, when the subject is already on antiviral therapy, that therapy can be continued or altered (e.g . by increasing the dosage and/or altering the type of therapy. In instances where the subject is not yet on an antiviral therapy, an appropriate antiviral therapy can be designed for the subject and administered.
  • the therapeutic methods can involve obtaining a biological sample from the subject (e.g . a blood or saliva sample), performing the methods described herein to determine whether or not HBV recrudescence is likely, and then exposing the subject to, or administering to the subject, antiviral therapy if it is determined that HBV recrudescence is likely.
  • a biological sample from the subject e.g . a blood or saliva sample
  • the therapeutic methods can involve sending a biological sample obtained from a subject to a laboratory to (i) conduct the methods of described herein to determine whether or not HBV recrudescence is likely, and (ii) provide the results of the method, wherein the results comprise a determination of whether HBV recrudescence is likely or unlikely, receiving the results from step (a); and then exposing the subject to, or administering to the subject, an antiviral therapy if the results comprise a determination that HBV recrudescence is likely.
  • Antiviral therapies appropriate for the treatment of HBV are well known in the art and include, for example, immunotherapy (e.g. interferon therapy, including pegylated IFN-a therapy), or nucleotide/nucleoside therapy (e.g. lamivudine (e.g. Epivir- HBV®), emtricitabine (e.g. Emtriva®) fovir dipivoxil (e.g. Hepsera®), entecavir (e.g. Baraclude®), telbivudine (e.g . Tyzeka®), clevudine (e.g. Levovir®) and tenofovir disoproxil (e.g. Viread®) .
  • immunotherapy e.g. interferon therapy, including pegylated IFN-a therapy
  • nucleotide/nucleoside therapy e.g. lamivudine (e.g. Epivir- HBV®), e
  • the present invention can be practiced in the field of predictive medicine for the purposes of predicting, detection or monitoring the recrudescence of HBV in a subject, and/or monitoring disease progression or response to therapy efficacy.
  • Saliva and/or blood samples were obtained from subjects enrolled in a clinical trial at Monash Medical Centre, Victoria.
  • the subjects enrolled in the trial included healthy subjects and subjects with chronic HBV infection and on therapy.
  • Subjects with chronic HBV infection were HBeAg negative and on nucleoside/nucleotide (NA) therapy (tenofovir, entecavir, adefovir or lamivudine), and met current APASL guidelines for consideration of antiviral cessation (uninterrupted nucleoside treatment for > 2years and undetectable serum HBV DNA on three separate occasions > 6 months apart (undetectable defined by a value ⁇ lower limit of detection using a sensitive commercial PCR assay).
  • NA nucleoside/nucleotide
  • the chronic HBV subjects also had normal serum alanine aminotransferase (ALT) levels and minimal to moderate liver fibrosis (defined as METAVIR liver fibrosis stage FO - F3 inclusive prior to initial NA therapy and/or transient liver elastogram (TLE) (Fibroscan®) ⁇ 9.6 kPa at screening).
  • ALT serum alanine aminotransferase
  • TLE transient liver elastogram
  • Exome sequencing was performed on a HiSeq3000 with 100 base pair, paired end sequencing and four samples per lane. This provided between 84 and 104 million reads per sample. On average, coverage was greater than lOOx. Reads were analysed with FastQC for quality
  • the codon context within the structure of the mutated codon was determined, i.e. the position of the SNV within the encoding triplet was determined, wherein the first position (read from 5' to 3') is referred to as MCI, the second position is referred to as MC2 and the third position is referred to as MC3;
  • a nine-base window was extracted from the surrounding genome sequence such that the sequence of three complete codons was obtained.
  • the direction of the gene was used for determining 5' and 3' directions, and for determining the correct strand of the nine bases.
  • the nine-base window was always reported according to the direction of the gene such that bases in the window around variants in genes on the reverse strand of the genome are reverse complimented in relation to the genome, but in the forward direction in relation to the gene. By convention, this context is always reported in the same strand of the gene. Positive strand genes will have codon context bases from the positive strand of the reference genome, and negative strand genes will have codon context bases from the negative strand of the reference genome;
  • c) motif searching was performed using known motifs for the fou r main deaminases to determine whether the variation was within such a motif and thus resulted from deaminase activity, with the four main deaminase motifs being as follows (wherein the underlined base corresponds to the targeted/mutated base, and the target motif to the right of the forward slash is the reverse compliment of the forward strand motif that is used for searching on the reverse strand of the gene) :
  • APOBEC3B - TCA/TGA SNVs attributable to AID, ADAR, APOBEC3G or APOBEC3B were identified.
  • the number of SNVs at particular positions within the mutated codon were also calculated, as were the number of SNVs that involved mutation of an A, T, C or G to another nucleotide.
  • saliva and blood samples from five healthy subjects were collected and analysed as described in Example 1.
  • the samples included multiple saliva samples taken from each single subject in the same day, with one blood and three saliva samples taken from each of the five subjects. Reference intervals for saliva samples were calculated as the mean +/- 2 standard deviations (2STDEV) .
  • the SNVs in 24 healthy subjects were assessed to identify the number of SNVs of various types in healthy individuals, so as to determine the total mutation burden (i.e. the total number of SNVs) and then calculate normal range intervals for various genetic indicators of deaminase activity.
  • these indicators included the percentage of SNVs at the targeted nucleotide of the AID motif; the percentage of SNVs at the targeted nucleotide of the ADAR motif; the percentage of SNVs at the targeted nucleotide of the APOBEC3G motif; the percentage of SNVs at the targeted nucleotide of the APOBEC3B motif; the percentage of SNVs at an MC-3 site; the percentage of SNVs that were mutations of an adenine; the percentage of SNVs that were mutations of a thymine; and the percentage of SNVs that were mutations of a cytosine.
  • the ratio of the percentage of SNVs that were mutations of a cytosine to the percentage of SNVs that were mutations of a guanine (C:G ratio); the ratio of the percentage of SNVs that were mutations of an adenine to the percentage of SNVs that were mutations of a thymine (A:T ratio) ; and the ratio of the percentage of SNVs that were mutations of an adenine or a thymine to the percentage of SNVs that were mutations of a guanine or cytosine (AT:GC ratio) were calculated.
  • the average and standard deviations for each of these values was determined, and the range interval calculated as the average +/- 2 standard deviations.
  • Table 3 provides the results of the study, and sets forth the normal range intervals for each of the genetic indicators of deaminase activity.
  • H P_3_1 300 3.67 8.00 16.33 4.67 37.00 23.33 16.67 30.67 1.05 1.40 0.67
  • HBV recrudescence e.g. serum HBV DNA levels and alanine aminotransferase (ALT) levels
  • ALT alanine aminotransferase
  • HBV recrudescence at least one genetic indicator of deaminase activity was outside the normal range interval (H - high or L - low compared to the normal range interval), suggesting that the subject had an impaired deaminase activity and an impaired deaminase-associated immune response.
  • H - high or L - low compared to the normal range interval
  • an aberrant percentage of SNVs at an ADAR motif was associated with HBV recrudescence.
  • dysregulation of ADAR activity e.g. editing
  • HBV recrudescence a result consistent with the teachings in the prior art.
  • HBV recrudescence included an aberrant percentage of SNVs at an MC-3 site (possibly an indicator of 'off-target' somatic hypermutation (SHM) which is associated with progression to oncogenesis), and an aberrant percentage of mutations of an adenine (possibly indicative of reduced ADAR or Pol-eta function and/or disrupted MMR).
  • SHM 'off-target' somatic hypermutation
  • a predicted test score was calculated wherein if any of the genetic indicators of deaminase activity were abnormal, a score of 1 was assigned, with the total then calculated. All subjects that cleared the infection had a test score of 0, while all subjects that experienced reactivation of HBV had a score of > 1. This results clearly demonstrate that the genetic indicators of deaminase activity can be used to predict the likelihood of HBV recrudescence in a subject should that subject terminate antiviral therapy.
  • HBV_2_1 Flare Flare Flare 521.00 H 3.84 5.57 L 19.96 3.65 36.66 17.66 13.44 31.29 1.20 1.31 0.45
  • HBV_2_8 Flare Flare Flare 561.00 H 3.74 5.35 L 23.71 H 4.46 38.86 15.69 L 15.33 30.66 1.25 1.02 0.45
  • HBV_2_3 Clear Clear 607.00 H 4.12 7.41 19.77 4.94 37.40 20.76 15.16 32.29 1.02 1.37 0.56
  • HBV_2_9 Clear Clear 527.00 H 5.12 5.69 19.54 4.55 36.81 16.70 14.04 35.29 1.04 1.19 0.44
  • HBV_2_4 Clear Clear 525.00 H 4.57 6.10 21.71 4.38 39.05 16.95 13.52 36.38 1.10 1.25 0.44
  • HBV_2_5 Clear Clear 558.00 H 4.66 5.91 18.10 4.48 36.92 19.89 15.05 33.69 1.07 1.32 0.54

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Abstract

This invention relates generally to methods of determining the likelihood of hepatitis B virus recrudescence in a subject. The likelihood of recrudescence can be predicted based on the number, percentage, ratio and/or type of mutations that might be attributed to one or more endogenous deaminases in the genomic nucleic acid of a subject with HBV infection, compared to the genomic nucleic acid of a healthy subject.

Description

TITLE OF THE INVENTION
"METHODS OF DETERMINING THE LIKELIHOOD OF HEPATITIS B VIRUS RECRUDESCENCE"
FIELD OF THE INVENTION
[0001] This invention relates generally to methods of determining the likelihood of hepatitis B virus recrudescence in a subject.
RELATED APPLICATIONS
[0002] This application claims priority to Australian Provisional Application No.
2017902147 entitled "Methods of determining the likelihood of Hepatitis B virus recrudescence" filed 6 June 2017, the contents of which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Hepatitis B virus (HBV) is a double-stranded enveloped virus of the
Hepadnaviridae family. The virus particle contains an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity. The outer envelope contains embedded proteins which are involved in viral binding of, and entry into, susceptible cells, predominantly hepatocytes of humans and other higher primates.
[0004] The outcomes of HBV infection are age-dependent and include asymptomatic infection, acute hepatitis B, chronic HBV infection, cirrhosis and hepatocellular carcinoma (HCC). Acute hepatitis B occurs in approximately 1% of perinatal infections, 10% of early childhood infections (children aged 1 -5 years) and 30% of late infections (people aged >5 years) . Fulminant hepatitis develops in 0.1 -0.6% of acute hepatitis cases, with mortality from fulminant hepatitis B being approximately 70%.
[0005] The development of chronic HBV infection is inversely related to the age of acquisition, occurring in approximately 80-90% of people infected perinatally, about 30% of children infected before the age of 6 years, and in <5% of infections occurring in otherwise healthy adults. The number of patients with chronic HBV infection is estimated to be 350-400 million worldwide, with more than one million deaths annually resulting from cirrhosis, liver failure and hepatocellular carcinoma. HBV infection is therefore a major global health problem.
[0006] Therapies currently available for the treatment of HBV infection can be classified into two main categories: immunomodulator and nucleoside/nucleotide analogues. Although the efficacy of interferon alpha (INFa), a representative immunomodulator, has been established by a numerous studies, the clinical application of INFa has been compromised by the low overall response rate, adverse side effects and high cost. Nucleoside/nucleotide analogs are therefore now predominately employed in HBV therapy, with several nucleosides/nucleotides in clinical use, including (e.g . lamivudine (e.g . Epivir-HBV®), emtricitabine (e.g . Emtriva®) fovir dipivoxil (e.g . Hepsera®), entecavir (e.g . Baraclude®), telbivudine (e.g. Tyzeka®), clevudine (e.g . Levovir®) and tenofovir disoproxil (e.g . Viread®) . These agents significantly suppress the replication of HBV DNA to a very low level, leading to more favorable clinical outcomes, including a significant reduction in serious liver disease.
[0007] All therapies, however, have some side effects and also significant costs associated with them. Accordingly, where possible, it is desirable to terminate antiviral therapy. However, to date it has been very difficult to predict whether any single subject will be able to terminate therapy without subsequent recrudescence. Thus, there is a need in the art for a means of identifying those subjects on antiviral therapy who are at risk of HBV recrudescence if that therapy is terminated, and those subjects who can safely terminate therapy. Indeed, there is a need for identifying any subject at risk of HBV recrudescence, whether or not that subject is on antiviral therapy and whether or not the subject has chronic HBV.
SUMMARY OF THE INVENTION
[0008] The present invention is predicated in part on the determination that the number, percentage, ratio and/or type of mutations that might be attributed to one or more endogenous dea minases in the genomic nucleic acid of a subject with HBV infection, compared to the genomic nucleic acid of a healthy subject, is predictive of the likelihood that the subject will experience HBV recrudescence. In particular, the type and number of single nucleotide variations (SNVs) in a subject can be used to measure one or more genetic indicators of deaminase activity. As demonstrated herein, when any one of these genetic indicators of deaminase activity is outside a predetermined normal range interval for genetic indicators of deaminase activity (either below or above the normal range interval), the subject is likely to experience HBV recrudescence. Without being bound by theory, it is postulated that the presence of at least one genetic indicator of deaminase activity outside the predetermined normal range interval for deaminase activity indicates that the deaminase-associated innate immune response in the subject is impaired or dysregulated, such that the subject is unlikely to be able to effectively control HBV replication and infection . Accordingly, the methods disclosed herein provide a means for accurately predicting whether a subject will experience HBV recrudescence. In particular embodiments, the methods are useful for determining the likelihood that a subject with chronic HBV infection can successfully terminate antiviral therapy without HBV recrudescence. The methods can also be extended to therapeutic applications, whereby a course of therapy is prescribed based on the information provided by the predictive methods described herein.
[0009] In one aspect, the present invention provides method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject, the method comprising : analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs); measuring one or more genetic indicators of endogenous deaminase activity (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genetic indicators of endogenous deaminase activity) based on the number and/or type of SNVs detected so as to obtain a sample profile of genetic indicators of endogenous deaminase activity; and determining the likelihood of HBV recrudescence based on a comparison between the sample profile and a reference profile of genetic indicators of endogenous deaminase activity.
[0010] In a particular aspect, provided is a method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject, the method comprising : analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs) : measuring one or more genetic indicators of endogenous deaminase activity (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genetic indicators of endogenous deaminase activity) based on the number and/or type of SNVs detected; and determining that HBV recrudescence is likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity, or determining that HBV recrudescence is not likely when no genetic indicators of endogenous deaminase activity are outside predetermined normal range interval for those genetic indicators of endogenous deaminase activity.
[0011] In a further particular aspect, the invention provides a method for determining the likelihood of HBV recrudescence in a subject, the method comprising : analysing the sequence of a nucleic acid molecule from a subject to detect SNVs; measuring one or more genetic indicators of endogenous deaminase activity (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genetic indicators of endogenous deaminase activity) based on the number and/or type of SNVs detected; assigning a score to each genetic indicator of endogenous deaminase activity that is outside a predetermined normal range interval for the genetic indicator of endogenous deaminase activity and adding each score to calculate a total score; and determining that HBV recrudescence is likely when the total score is equal to or above a threshold score; or determining that HBV recrudescence is not likely when the total score is less than a threshold score.
[0012] In particular examples, the one or more genetic indicators of endogenous deaminase activity is selected from among the percentage of single nucleotide variations (SNVs) that are at a deaminase motif (optionally also specifying the type of mutation and/or codon context) ; the percentage of SNVs at MC-3 sites; the percentage of SNVs that include mutation of an adenine nucleotide; the percentage of SNVs that include mutation of a thymine nucleotide; the percentage of SNVs that include mutation of a cytosine nucleotide; the percentage of SNVs that include mutation of a guanine nucleotide; the ratio of the percentage of SNVs that include mutation of a cytosine nucleotide to the percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio); the ratio of the percentage of SNVs that include mutation of an adenine nucleotide to the percentage of SNVs that include mutation of a thymine nucleotide (A:T ratio); the ratio of the percentage of SNVs that include mutation of an adenine or a thymine nucleotide to the percentage of SNVs that include mutation of a cytosine or a guanine nucleotide (AT:GC ratio).
[0013] In some embodiments, the endogenous deaminase is selected from among activation-induced cytosine deaminase (AID), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 1 cytosine deaminase (AP0BEC1), AP0BEC3A, AP0BEC3B, AP0BEC3C, AP0BEC3D, AP0BEC3F, AP0BEC3G, AP0BEC3H and an adenosine deaminase acting on RNA (ADAR).
[0014] In a particular embodiment, the one or more genetic indicators of endogenous deaminase activity include the number or percentage of SNVs at an ADAR motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an AP0BEC3G motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an AP0BEC3B site (optionally also specifying the type of mutation and/or codon context); the number or percentage of the percentage of SNVs at MC-3 sites; and/or the number or percentage of SNVs resulting from mutation of an adenine nucleotide. In still particular embodiments, the methods comprise assessing one or more genetic indicators of endogenous deaminase activity
[0015] In embodiments where the one or more genetic indicators of endogenous deaminase activity includes the percentage of single nucleotide variations (SNVs) at a deaminase motif, exemplary deaminase motifs include an AID motif selected from among motifs comprising the nucleic acid sequence WRC/GYW and WRCG/CGYW (wherein the underlined nucleotide is mutated) ; an ADAR motif comprising the nucleic acid sequence WA/TW; WAY/RTW; WTAW/WTAW; RAWA/TWTY; AAC/GTT; AA /TTT; ACA/TGT; CCA/TGG; CGA TCG; and CTA/JAG (wherein the underlined nucleotide is mutated); an APOBEC3G motif selected from among motifs comprising the nucleic acid sequence CC/CC, CG/CG, CG/CG, TCG/CGA, CCG/CGG and CGG/CCG (wherein the underlined nucleotide is mutated) ; an APOBEC3B motif selected from among motifs comprising the nucleic acid sequence TCA/TGA, TC/GA and TCW/WGA (wherein the underlined nucleotide is mutated) ; an APOBEC3H motif comprising the nucleic acid sequence TCW/WGA; an APOBEC1 motif selected from among motifs comprising the nucleic acid sequence TG/CA and GG/CC; and an APOBEC3A or APOBEC3F motif selected from among motifs comprising the nucleic acid sequence TC/GA and TCW/WGA.
[0016] In some examples of the methods described above and herein, the one or more genetic indicators of deaminase activity that are assessed comprise a genetic indicator of ADAR activity, a genetic indicator of APOBEC3B activity and/or a genetic indicator of APOBEC3G activity. In still particular embodiments, the methods comprise assessing at least one or more genetic indicators of ADAR activity and at least one or more genetic indicators of APOBEC3B activity; at least one or more genetic indicators of ADAR activity and at least one or more genetic indicators of APOBEC3G activity; at least one or more genetic indicators of APOBEC3B activity and at least one or more genetic indicators of APOBEC3G activity; or at least one or more genetic indicators of ADAR activity, at least one or more genetic indicators of APOBEC3B activity and at least one or more genetic indicators of APOBEC3G activity.
[0017] For example, a genetic indicator of ADAR activity may be selected from among the number or percentage of SNVs resulting from a mutation of A and the number or percentage of SNVs that are within an ADAR motif (e.g. the number or percentage of total SNVs that are within the motif WA/TW; number or percentage of total SNVs that are within the motif AAA/TTT; number or percentage of SNVs within the motif ACA/TGT that are A>C mutations; number or percentage of T>C mutations within the motif AAC/GTT that are at MC2 sites; number or percentage of SNVs within the motif CCA/TGG that are T>A; number or percentage of A>G mutations within the motif CGA/TCG that are at MCI sites; number or percentage of SNVs within the motif CTA/TAG that are T>A; number or percentage of all SNVs that are transitions at the WA/TW motif; number or percentage of SNVs within the WA/TW motif that are at MCI sites; number or percentage of SNVs within the WA/TW motif that are at MC2 sites; number or percentage of SNVs within the WA/TW motif that are at MC3 sites; number or percentage of A>G mutations within the WA/TW motif that are at MCI sites; number or percentage of A>G mutations within the WA/TW motif that are at MC2 sites; number or percentage of A>G mutations within the WA/TW motif that are at MC3 sites; number or percentage of T>C mutations within the WA/TW motif that are at MCI sites; number or percentage of T>C mutations within the WA/TW motif that are at MC2 sites; number or percentage of T>C mutations within the WA/TW motif that are at MC3 sites; number or percentage of SNVs within the WA motif that are A>G mutations; number or percentage of SNVs within the WA motif that are A>C mutations; number or percentage of SNVs within the WA motif that are A>T mutations; number or percentage of SNVs within the TW motif that are T>C mutations; number or percentage of SNVs within the TW motif that are T>G mutations; number or percentage of SNVs within the TW motif that are T>A mutations; or the number or percentage of SNVs within the WA/TW motif that are transitions).
[0018] A genetic indicator of APOBEC3B activity can be the number or percentage of SNVs that are within an APOBEC3B motif (e.g. the number or percentage of mutations at motifs TCA/TGA, TC/GA and/or TCW/WGA; number or percentage of all SNVs that are transitions at the TCW/WGA motif; number or percentage of SNVs within the TCW/WGA motif that are at MCI sites; number or percentage of SNVs within the TCW/WGA motif that are at MC2 sites; number or percentage of SNVs within the TCW/WGA motif that are at MC3 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MCI sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MCI sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of SNVs within the TCW motif that are C>T mutations; number or percentage of SNVs within the TCW motif that are C>A mutations; number or percentage of SNVs within the TCW motif that are C>G mutations; number or percentage of SNVs within the WGA motif that are G>A mutations; number or percentage of SNVs within the WGA motif that are G>T mutations; number or percentage of SNVs within the WGA motif that are G>C mutations; number or percentage of SNVs within the TCW/WGA motif that are transitions).
[0019] In other examples, a genetic indicator of APOBEC3G activity is the number or percentage of SNVs that are within an APOBEC3G motif (e.g. the number or percentage of mutations at motifs CC/GG, CG/CG, CG/CG, TCG/CGA, and/or CGG/CCGA; the number or percentage of C to T mutations at the CC motif; the number or percentage of C to T mutations at the CG motif; the number or percentage of G to A mutations at the CG motif; the number or percentage of mutations within the motif TCGA/TCGA that are G to T; number or percentage of all SNVs that are transitions at the CC/GG motif; number or percentage of SNVs within the CC/GG motif that are at MCI sites; number or percentage of SNVs within the CC/GG motif that are at MC2 sites; number or percentage of SNVs within the CC/GG motif that are at MC3 sites; number or percentage of C>T mutations within the CC/GG motif that are at MCI sites; number or percentage of C>T mutations within the CC/GG motif that are at MC2 sites; number or percentage of C>T mutations within the CC/GG motif that are at MC3 sites; number or percentage of G>A mutations within the CC/GG motif that are at MCI sites; number or percentage of G>A mutations within the CC/GG motif that are at MC2 sites; number or percentage of G>A mutations within the CC/GG motif that are at MC3 sites; number or percentage of SNVs within the CC motif that are C>T mutations; number or percentage of SNVs within the CC motif that are C>A mutations; number or percentage of SNVs within the CC motif that are C>G mutations; number or percentage of SNVs within the GG motif that are G>A mutations; number or percentage of SNVs within the GG motif that are G>T mutations; number or percentage of SNVs within the GG motif that are G>C mutations; number or percentage of SNVs within the CC/GG motif that are transitions).
[0020] In certain embodiments, the SNVs do not include known single nucleotide polymorphisms.
[0021] In some examples, the nucleic acid molecule has been obtained from a blood or saliva sample from the subject. In further examples, the methods further comprise obtaining a biological sample (such as a blood or saliva sample) from the subject and extracting the nucleic acid molecule.
[0022] In particular embodiments, the subject has chronic HBV infection. In some of the same and other embodiments, the subject is on antiviral therapy. Antiviral therapy can include, for example, nucleoside/nucleotide therapy (e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy) or immunotherapy (e.g. interferon-alpha (IFN-a) therapy).
[0023] The methods of the present invention may further comprise providing a recommendation to the subject to continue antiviral therapy if it is determined that HBV recrudescence is likely, or further comprise providing a recommendation to the subject to terminate antiviral therapy if it is determined that HBV recrudescence is not likely.
[0024] Another aspect of the present invention relates to use of an antiviral therapy for treating HBV infection in a subject, wherein the subject is exposed to the antiviral therapy on the basis of a determination that HBV recrudescence is likely as described above and herein. Antiviral therapy can include, for example, nucleoside/nucleotide therapy (e.g. lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy) or immunotherapy (e.g . interferon-alpha (IFN-a) therapy). [0025] In a further aspect, the invention relates to a method for treating HBV infection in a subject, comprising : performing the method described above a nd herein to determine whether HBV recrudescence is likely, and exposing the subject to an antiviral therapy if it is determined that HBV recrudescence is likely.
[0026] In another aspect, the present invention is directed to a method for treating HBV infection in a subject, comprising : (a) sending a biological sample obtained from a subject to a laboratory to (i) conduct the method of described above and herein to determine whether HBV recrudescence is likely, and (ii) provide the results of the method, wherein the results comprise a determination of whether HBV recrudescence is likely or unlikely; (b) receiving the results from step (a); and (c) exposing the subject to an antiviral therapy if the results comprise a determination that HBV recrudescence is likely.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0028] The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "a genetic indicator" means one genetic indicator or more than one genetic indicator.
[0029] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
[0030] The term "about", as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range {e.g. , 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about".
[0031] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a subject or patient. Suitably, the biological sample is selected from any part of a patient's body, including, but not limited to bodily fluids such as saliva or blood, tissue, cells, hair, skin and nails.
[0032] As used herein, the term "codon context" with reference to a mutation refers to the nucleotide position within a codon at which the mutation occurs. For the purposes of the present invention, the nucleotide positions within a mutated codon (MC; i.e., a codon containing the mutation) are annotated MC-1, MC-2 and MC-3, and refer to the first, second and third nucleotide positions, respectively, when the sequence of the codon is read 5' to 3'. Accordingly, the phrase "determining the codon context of a mutation" or similar phrase means determining at which nucleotide position within the mutated codon the mutation occurs, i.e. , MC-1, MC-2 or MC-3.
[0033] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.
[0034] The term "control subject", as used in the context of the present invention, may refer to a subject known to be affected with HBV (positive control), or to a subject known to be not affected or diagnosed with HBV (negative control). It should be noted that a control subject that is known to be healthy, i.e. , not having an HBV infection, may possibly suffer from another disease not tested/known . It is also understood that control subjects and healthy controls include data obtained and used as a standard, i.e., it can be used over and over again for multiple different subjects. In other words, for example, when comparing a subject sample to a control sample, the data from the control sample could have been obtained in a different set of experiments, for example, it could be an average obtained from a number of healthy subjects and not actually obtained at the time the data for the subject was obtained. [0035] The term "correlating" generally refers to determining a relationship between one type of data with another or with a state. In various embodiments, correlating deaminase activity with the likelihood that a subject will experience HBV recrudescence comprises assessing genetic indicators of deaminase activity in a subject and comparing the levels of these indicators to genetic indicators of deaminase activity in persons known to be free of HBV infection or to predetermined normal range intervals for genetic indicators of deaminase activity.
[0036] By "gene" is meant a unit of inheritance that occupies a specific locus on a genome and comprises transcriptional and/or translational regulatory sequences a nd/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).
[0037] As used herein, a "genetic indicator of deaminase activity" refers to a number, percentage, ratio and/or type of a single nucleotide variation (SNV) that may be reflective of the activity of one or more endogenous deaminases. Exemplary genetic indicators of deaminase activity include, but are not limited to, the number or percentage of SNVs at a deaminase motif; the number or percentage of SNVs at MC-3 sites; the number or percentage of SNVs that include mutation of an adenine nucleotide; the number or percentage of SNVs that include mutation of a thymine nucleotide; the number or percentage of SNVs that include mutation of a cytosine nucleotide; the number or percentage of SNVs that include mutation of a guanine nucleotide; the ratio of the number or percentage of SNVs that include mutation of a cytosine nucleotide to the number or percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio); the ratio of the number or percentage of SNVs that include mutation of an adenine nucleotide to the number or percentage of SNVs that include mutation of a thymine nucleotide (A:T ratio); the ratio of the number or percentage of SNVs that include mutation of an adenine or a thymine nucleotide to the number or percentage of SNVs that include mutation of a cytosine or a guanine nucleotide (AT:GC ratio).
[0038] As used herein, the term "likelihood" or grammatical variations is used as a measure of whether HBV recrudescence will occur, and of whether subjects with a genetic indicator of deaminase activity that is outside a predetermined normal range interval, or with a particular sample profile of genetic indicators, will experience HBV recrudescence based on a given mathematical model. An increased likelihood for example may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased likelihood or risk that a subject will experience HBV recrudescence may be expressed as determining whether any genetic indicators of deaminase activity are identified as being outside the normal range interval (as taught herein) and placing the test subject in an "increased likelihood or risk" category, based upon previous population studies.
[0039] In some embodiments, the methods comprise comparing a score based on the number of genetic indicators of deaminase activity that are outside a predetermined normal range interval to a "threshold score". The threshold score is one that provides an acceptable ability to predict the likelihood of HBV recrudescence in a subject, and can be determined by those skilled in the art using any acceptable means. In some examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population has a first condition or risk and a second population has a second condition or risk (called arbitrarily, for example, "controlled infection" and "HBV recrudescence", or "low risk" and "high risk").
[0040] A distribution of number of number of genetic indicators of deaminase activity that are outside a predetermined normal range interval in subjects who experience disease progression and in subjects who do not experience disease progression may overlap. Under such conditions, a test does not absolutely distinguish between disease progression and disease stability or regression with 100% accuracy. A threshold is selected, above which the test is considered to be "positive" and below which the test is considered to be "negative." The area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, for example, Hanley et a/, Radiology 143 : 29-36 (1982)). The term "area under the curve" or "AUC" refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest. ROC curves are useful for plotting the performance of a particular feature in disting uishing or discriminating between two populations (e.g., subjects with disease progression and subjects without disease progression). Typically, the feature data across the entire population (e.g., the cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data are calculated. The sensitivity is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases. The specificity is determined by counting the number of controls below the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated in cases compared to controls, this definition also applies to scenarios in which a feature is lower in cases compared to the controls (in such a scenario, samples below the value for that feature would be counted). ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to produce a single value, and this single value can be plotted in a ROC curve. Additionally, any combination of multiple features (e.g. , one or more other epigenetic markers), in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test. The ROC curve is the plot of the sensitivity of a test against the specificity of the test, where sensitivity is traditionally presented on the vertical axis and specificity is traditionally presented on the horizontal axis. Thus, "AUC ROC values" are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. An AUC ROC value may be thought of as equivalent to the Mann - Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.
[0041] The term "diagnosis" as used in the context of the present invention refers to the process of determining the likelihood of HBV recrudescence. An assessment of genetic indicators of deaminase activity in a nucleic acid present in a biological sample from a subject is used to determine the likelihood of HBV recrudescence in a subject.
[0042] As used herein "disease progression" refers to a worsening of a disease over time. Disease progression can be associated with or measured by an increase or worsening of one or more symptoms or markers associated with the disease (e.g. an increase in serum alanine aminotransferase levels) . In instances where the disease is an infection, disease progression can also be associated with or measured by an increase in viral, bacterial or fungal load (e.g. an increase in detectable serum HBV).
[0043] As used herein, a "healthy" subject is a subject that does not have HBV infection.
[0044] As used herein, "level" with reference to a SNV or genetic indicator of deaminase activity refers to the number, percentage, amount or ratio of SNV or genetic indicator of deaminase activity.
[0045] As used herein, a "mutation type" refers to the specific nucleotide substitution that comprises the mutation, and is selected from among C to T, C to A, C to G, G to T, G to A, G to C, A to T, A to C, A to G, T to A, T to C and T to G mutations. Thus, for example, a mutation type of C to T refers to a mutation in which the targeted or mutated nucleotide C is replaced with the substituting nucleotide T. [0046] The "nucleic acid" as used herein designates DNA, cDNA, mRNA, RNA, rRNA or cRNA. The term typically refers to polynucleotides greater than 30 nucleotide residues in length.
[0047] As used herein, a "predetermined normal range interval" refers to a range of values, with an upper and lower limit, for a genetic indicator of deaminase activity that are reflective of a healthy or non-disease state. The predetermined normal range interval can be determined by assessing a genetic indicator of deaminase activity in two or more non-diseased or healthy subjects (e.g. subjects that do not have HBV infection and/or who do not have any known infection or disease). A normal range interval for the genetic indicator is then calculated to set the upper and lower limits of what would be considered normal values for that indicator, e.g. values that would reflect a normal deaminase activity in healthy individuals. In a particular example, the normal range interval is calculated by measuring the average plus or minus 2 standard deviations, whereby the lower limit of the range interval is the average minus 2 standard deviations and the upper limit of the range interval is the average plus 2 standard deviations. In other examples, less than or more than 2 standard deviations is used to set the upper and lower limits of the interval, such as 1, 1.5, 2.5, 3, 3.5 or more standard deviations. In still further examples, the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves. The subjects used to determine the predetermined normal range interval can be of any age, sex or background, or may be of a particular age, sex, ethnic background or other subpopulation. Thus, in some embodiments, two or more predetermined normal range intervals can be calculated for the same genetic indicator of deaminase activity, whereby each range interval is specific for a particular subpopulation, e.g. a particular sex, age group, ethnic background and/or other subpopulation.
[0048] As used herein, "recrudescence" refers to the reappearance of an HBV infection after it has been quiescent i.e. after a period of latency or relative inactivity. Recrudescence of HBV can be assessed by direct detection of HBV DNA, RNA and/or antigen, and/or by indirect detection of other markers, such as serum alanine aminotransferase (ALT) levels. Recrudescence encompasses both short- and long-term reappearance of infection, sometimes also referred to as reactivation and relapse. The terms "reactivation" or "reactivate" refer to a sudden increase in HBV replication in a subject, and typically is confirmed when there is detectable serum HBV DNA of less than 2000 IU/mL and/or serum ALT of 1.2-5 x upper limit of normal (ULN) . As used herein, "relapse" refers to a sustained or increased reactivation of HBV. Typically, detectable serum HBV DNA of greater than 2000 IU/ml, and/or serum ALT of greater than 5 x ULN for 16 weeks or more, or serum ALT of greater than 10 x ULN for 8 weeks or more, is indicative of HBV relapse.
[0049] As used herein, a "reference profile of genetic indicators of deaminase activity" provides an analysis in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genetic indictors of deaminase activity are evaluated. The reference profile is associated with a defined risk or likelihood of HBV recrudescence, such as "no risk/likelihood", negligible risk/likelihood", "low risk/likelihood", "medium risk/likelihood" or "high risk/likelihood". Accordingly, by virtue of comparison to the reference profile, the risk of a subject experiencing HBV recrudescence can be determined, i.e. by obtaining the profile of genetic indicators of deaminase activity for the subject (i.e. the sample profile) and comparing it to the reference profile.
[0050] As used herein, "single nucleotide variation" refers to a variation occurring in the sequence of a nucleic acid molecule (e.g. a subject nucleic acid molecule) compared to another nucleic acid molecule (e.g. a reference nucleic acid molecule or sequence), wherein the variation is a difference in the identity of a single nucleotide (e.g. A, T, C or G).
[0051] The term "somatic mutation" refers to a mutation in the DNA of somatic cells (i.e., not germ cells), occurring after conception. "Somatic mutagenesis" therefore refers to the process by which somatic mutations occur.
[0052] The terms "subject", "individual" or "patient", used interchangeably herein, refer to any animal subject, particularly a mammalian subject. By way of an illustrative example, suitable subjects are humans. In some embodiments, the subject presents with clinical signs of a condition as defined herein. As used herein, the term "clinical sign", or simply "sign", refers to objective evidence of a disease present in a subject. Symptoms and/or signs associated with diseases referred to herein and the evaluation of such signs are routine and known in the art. Examples of signs of disease vary depending upon the disease. Signs of a HBV infection may include detection of HBV DNA, HBV antigen and/or elevated serum ALT levels. Typically, whether a subject has a disease, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the disease.
[0053] As used herein, the terms "targeted somatic mutagenesis" and "TSM" refer to the process of somatic mutagenesis resulting from one or more mutagenic agents, wherein mutagenesis occurs at a targeted nucleotide within a motif, the targeted nucleotide is present at a particular position within a codon (e.g. , the first, second or third position of the mutated codon reading from 5' to 3', annotated MC-1, MC-2 and MC- 3, respectively), and the targeted nucleotide is mutated to a particular substituting nucleotide (i.e., the mutation is of a particular mutation type, e.g., C to T, not C to A or C to G) . Thus, a determination that TSM is occurring requires analysis of the type of mutation (e.g., C to T), the motif at which the mutation occurs (e.g. , WRC) and codon context of the mutation, i.e., the position within the codon at which the mutation occurs (e.g., MC-1, MC-2 or MC-3). "Targeted somatic mutagen" therefore refers to mutation resulting from TSM.
[0054] The terms "treat" and "treating" as used herein, unless otherwise indicated, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, either partially or completely, ameliorate or slow down (lessen) the targeted condition or disorder (e.g., HBV infection), or one or more symptom associated therewith. The terms are also used herein to denote inhibiting the replication of HBV and/or alleviating the effects of HBV. Those in need of treatment include those diagnosed with HBV, those suspected of having HBV or those at risk of getting HBV. Hence, the subject to be treated herein may have been diagnosed as having the HBV or may be at risk of contracting HBV. In some embodiments, treatment refers to the eradication, removal, modification, or control of HBV infection that results from the administration of one or more therapeutic agents according to the methods of the invention. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating.
[0055] Those skilled in the art will appreciate that the aspects and embodiments described herein are susceptible to variations and modifications other than those specifically described . It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
2. Abbreviations
[0056] The following abbreviations are used throughout the application :
ADAR = adenosine deaminases acting on RNA
AID = activation-induced cytosine deaminase
APOBEC = apolipoprotein B mRNA-editing enzyme, catalytic
polypeptide-like (APOBEC) cytosine deaminases
ds = double stranded
h = hours
min = minutes
NTS = non-transcribed strand
SHM = somatic hypermutation SNV = single nucleotide variation
ss = single stranded
TS = transcribed strand
TSM = targeted somatic mutation
TABLE 1
NUCLEOTIDE SYMBOLS
Figure imgf000017_0001
[0057] The present invention is predicated in part on the unexpected finding that the number, percentage, ratio and/or type of mutations that may be reflective of the activity of one or more endogenous deaminases in the genomic nucleic acid of a subject with HBV infection, compared to the genomic nucleic acid of a healthy subject, is predictive of the likelihood of HBV recrudescence in that subject. The present invention encompasses methods for identifying these changes in endogenous deaminase activity as well as methods for determining the likelihood of HBV recrudescence in a subject by detecting these changes.
3. Endogenous deaminases
[0058] Endogenous deaminases are known to be involved in somatic mutagenesis, including somatic hypermutation and class switch recombination of immunoglobulin genes in B cells. In addition, endogenous deaminases are key factors in RNA editing and innate immunity. For example, a number of deaminases have been shown to be involved in editing of viral RNA and DNA as a means to inhibit or reduce viral replication, and have also been implicated in activating other factors of innate immune response to combat viral infection, including (see e.g. Samuel (2012) Curr Top Microbiol Immunol. 353; Vieira and Soares (2013) BioMed Research International, 683095 ; He et al. (2015) Mol Med Rep. 12(5) : 6405-6414). Conversely, in some instances, deaminases appear to have a proviral function (see e.g. Samuel (2011) Virology 411 : 180-193).
[0059] Endogenous deaminases include, for example, adenosine deaminases such as adenosine deaminases acting on RNA (ADAR), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytosine deaminases, and activation- induced cytosine deaminase (AID), and error-prone DNA polymerases such as DNA polymerase eta. These endogenous deaminases preferentially target specific motifs in the nucleic acid. Moreover, there can be both a strand bias and codon context associated with mutation events resulting from these deaminases, as described in, for example, WO 2014/066955 and Lindley et al. (2016) Cancer Med. 2016 Sep; 5(9) : 2629-2640. This specific type of mutation event has been termed targeted somatic mutation (TSM). More specifically, TSM refers to the process of somatic mutagenesis resulting from one or more mutagenic agents, such as a deaminase, wherein mutagenesis occurs at a targeted nucleotide within a motif, the targeted nucleotide is present at a particular position within a codon (e.g., the first, second or third position of the mutated codon reading from 5' to 3', annotated MC-1, MC-2 and MC-3, respectively), and the targeted nucleotide is mutated to a particular substituting nucleotide (i.e. , the mutation is of a particular mutation type, e.g. , C to T, not C to A or C to G). Thus, a determination that TSM is occurring requires analysis of the type of mutation (e.g., C to T), the motif at which the mutation occurs (e.g., WRC) and codon context of the mutation, i.e., the position within the codon at which the mutation occurs (e.g. , MC-1, MC-2 or MC-3).
[0060] Activation-induced cytosine deaminase (AID) is an important enzyme in adaptive immunity, involved in somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes in B cells. AID triggers SHM by deaminating cytosines to uracils (C to U) to diversify the immunoglobulin variable region genes (VDJ) and create new antigen-binding sites. If unrepaired, the deamination of C to U by AID gives rise to C to T somatic mutations in DNA. AID has been shown to be involved in the innate antiviral immune response, including editing the DNA and RNA of various viruses such as HBV (see e.g . He et al. (2015) Mol Med Rep. 12(5) : 6405-6414, Liang et al. Proc Natl Acad Sci U S A. 110(6) : 2246-51).
[0061] In addition to AID, the human genome encodes several homologous
APOBEC cytosine deaminases that are known to be involved in innate immunity and RNA editing (Smith et al. (2012) Semin. Cell. Dev. Biol. 23: 258-268). In humans, at least AP0BEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H are involved in providing innate immunity and/or cellular mRNA editing. These APOBEC deaminases are also involved in the innate immune response, editing the DNA and RNA of various viruses (including HBV) and disrupting viral replication (see e.g. Turelli et al. (2004) Science 303 : 1829; Suspene et al. (2005) Proc Natl Acad Sci USA 102(23) : 8321-8326; Nguyen et al. (2007) J. Virol. 81 :4465 - 44472; Kock and Blum (2008) J Gen. Virol. 89: 1184-1191 ; He et al. (2015) Mol Med Rep. 12(5) : 6405-6414).
[0062] Double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes, are responsible for the type of RNA editing that is most prevalent in higher eukaryotes, i.e. conversion of adenosine residues into inosine (A-to-I editing). ADAR is an enzyme that is encoded by the ADAR gene in humans. The ADAR1 enzyme destabilizes dsRNA through conversion of adenosine to inosine. The ADAR1 enzyme modifies cellular and viral RNA, including coding and noncoding RNAs. ADAR1 is an RNA editing enzyme, required for hematopoiesis. ADAR1+/" chimeric embryos die before embryonic day 14 with defects in the hematopoietic system. Regulated levels of ADAR1 expression are critical for embryonic erythropoiesis in the liver. Mutations in the ADAR gene have been associated with dyschromatosis symmetrica hereditaria. Alternate transcriptional splice variants, encoding different isoforms, have been characterized.
[0063] Like other deaminases, ADARs have been implicated in the innate immune response to viruses. However, their precise role is in many instances not well defined, and in some examples, there appears to be both and antiviral and proviral role for ADARs (see e.g. Samuel (2011) Virology 411(2) : 180-93).
4. Genetic indicators of endogenous deaminase activity
[0064] The activity of any one or more deaminases can be assessed as described herein by detecting mutations that are reflective of deaminase activity, such as ADAR, APOBEC3B and/or APOBEC3G activity. In particular, single nucleotide variations (SNVs) that may be the result of deaminase activity are detected . The number or percentage of these SNVs, and/or the relationship (e.g. the ratio) between these SNVs and other nucleotides in the nucleic acid molecule, may be indicative of deaminase activity. Accordingly, based on the type and/or number of the SNVs, genetic indicators of deaminase activity can be identified.
[0065] Genetic indicators of deaminase activity for use in the methods of the present invention include those described above and below. Exemplary of such indicators are, for example, the number or percentage of SNVs at a deaminase motif, optionally specified by mutation type and/or MC site (e.g. the number or percentage of C>T mutations within the AID motif WRC/GYW that are at MCI sites; the number or percentage of G>A mutations within the AID motif WRC/GYW that are at C2 sites; the number or percentage of C>T mutations within the motif ACA/TGT that are at MC2 sites; the number or percentage of G>A mutations within the motif CCA/TGG that are at MCI sites; the number or percentage of G>A mutations within the motif GCT/AGC that are at MC2 sites; the number or percentage of mutations within the APOBEC3B (A3B) motif TCW/WGA that are at MC2 sites; the number or percentage of mutations within the APOBEC3G (A3G) motif TCGA/TCGA that are G>T; the number or percentage of T>C mutations within the ADAR motif AAC/GTT that are at MC2 sites; the number or percentage of total mutations that are within the ADAR motif AAA/ITT; the number or percentage of mutations within the ADAR motif ACA/TGT that are A>C mutations; the number or percentage of mutations within the motif CCA/TGG that are T>A; the number or percentage of A>G mutations within the motif CGA/TCG that are at MCI sites; and the number or percentage of mutations within the motif CT A/TAG that are T>A); the number or percentage of SNVs at MC-3 sites; the number or percentage of SNVs that include mutation of an adenine nucleotide; the number or percentage of SNVs that include mutation of a thymine nucleotide; the number or percentage of SNVs that include mutation of a cytosine nucleotide; the number or percentage of SNVs that include mutation of a guanine nucleotide; the ratio of the number or percentage of SNVs that include mutation of a cytosine nucleotide to the number or percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio); the ratio of the number or percentage of SNVs that include mutation of an adenine nucleotide to the number or percentage of SNVs that include mutation of a thymine nucleotide (A:T ratio); the ratio of the number or percentage of SNVs that include mutation of an adenine or a thymine nucleotide to the number or percentage of SNVs that include mutation of a cytosine or a guanine nucleotide (AT:GC ratio).
4.1 SNVs in deaminase motifs
[0066] In some instances the methods of the present invention comprise detecting SNVs at the targeted nucleotide in a deaminase motif. The total number and/or percentage of total SNVs at a given motif, or at multiple motifs of a particular deaminase, is then calculated . The motif may be any one or more motifs targeted by any one or more endogenous deaminases, including AID, ADAR, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and/or APOBEC3H . The motifs may be on the non-transcribed and/or transcribed strand of the nucleic acid. In particular examples, the motifs are on the non-transcribed strand (NTS) of the nucleic acid. [0067] Exemplary AID motifs include, for example, a motif comprising the nucleic acid sequence WRC/GYW (where R = A/G, W = A/T, Y = T/C and wherein the underlined nucleotide is mutated); a motif comprising the nucleic acid sequence WA (where W = A/T and wherein the underlined nucleotide is mutated); and a motif comprising the nucleic acid sequence WRCG/CGYW (where R = A/G, W = A/T, Y = T/C and wherein the underlined nucleotide is mutated).
[0068] Exemplary motifs utilized by AP0BEC3G include the motif comprising the nucleic acid sequence CC/GG; the motif comprising the nucleic acid sequence CG/CG; and the motif comprising the nucleic acid sequence CG/CG (wherein the underlined nucleotide is mutated). Other motifs that can be targeted by AP0BEC3G include the motif comprising the nucleic acid sequence TCG/CGA; the motif comprising the nucleic acid sequence CCG; and the motif comprising the nucleic acid sequence CGG/CCG.
[0069] An exemplary AP0BEC3B motif comprises the nucleic acid sequence
TCA/TGA (wherein the underlined nucleotide is mutated); TC/GA (wherein the underlined nucleotide is mutated); or TCW/WGA (where W = A/T and wherein the underlined nucleotide is mutated).
[0070] A non-limiting example of a motif targeted by AP0BEC3H is one comprising a nucleic acid sequence GA/TC (wherein the underlined nucleotide is mutated).
[0071] Exemplary AP0BEC1 motifs include those comprising the nucleic acid sequence TG/CA or GG/CC (wherein the underlined nucleotide is mutated).
[0072] AP0BEC3A and AP0BEC3F have been shown to target a motif comprising the nucleic acid sequence TC/GA (wherein the underlined nucleotide is mutated) or TCW/WGA (where W = A/T and wherein the underlined nucleotide is mutated).
[0073] Exemplary of the motifs targeted by ADAR is the motif comprising the nucleic acid sequence WA/TW (wherein the underlined nucleotide is mutated). Other exemplary motifs include the motif comprising the nucleic acid sequence WAY/RTW; the motif comprising the nucleic acid sequence RAWA/TWTY; the motif comprising the nucleic acid sequence WTAW/WTAW; the motif comprising the nucleic acid sequence SARA/TYTS; the motif comprising the nucleic acid sequence AAC/GTT; the motif comprising the nucleic acid sequence AAA/TTT; the motif comprising the nucleic acid sequence ACA TGT; the motif comprising the nucleic acid sequence CCA/TGG; the motif comprising the nucleic acid sequence CGA/TCG; and the motif comprising the nucleic acid sequence CTA/TAG. [0074] In a particular embodiment, the percentage of SNVs in an AID motif
(e.g. WRC/GYW), ADAR motif (e.g. WA/TW), APOBEC3G (e.g. CC/GG) and/or APOBEC3B motif (e.g. TCA/TGA) is determined.
[0075] Typically, the methods of the present disclosure involve the detection of any mutation at the targeted nucleotide, e.g. an A, C or T off a G. Moreover, the methods typically involve the detection of a mutation when the targeted nucleotide is at any position within the codon (i.e. at MC-1, MC-2 or MC-3). Thus, a SNV at a deaminase motif is found to be present when there is any mutation at the targeted nucleotide within the motif and when the targeted nucleotide is at any position within the codon (as described in Examples 1-5, below).
[0076] In other embodiments, detection of a SNV at a deaminase motif involves a further assessment of the type of a mutation and/or the codon context of a mutation before it is determined whether a SNV is attributable to deaminase activity. For example, in some embodiments, a SNV at a deaminase motif is detected when there is a C to T mutation at the WRC AID motif; a G to A mutation at the GYW AID motif; an A to G mutation at the WA AID motif; an A to T mutation at the WA ADAR motif; a C to T mutation at the CC AP0BEC3G motif; a C to T mutation at the CG AP0BEC3G motif; a G to A mutation at the CG AP0BEC3G motif; a G to A mutation at the GA AP0BEC3H motif; a G to A mutation at the TG AP0BEC1 motif; or a G to T mutation at the GG AP0BEC1 motif; C to T mutations within the AID motif WRC/GYW that are at MCI sites; G to A mutations within the AID motif WRC/GYW that are at MC2 sites; C to T mutations within the motif ACA/TGT that are at MC2 sites; G to A mutations within the motif CCA/TGG that are at MCI sites; G to A mutations within the motif GCT/AGC that are at MC2 sites; mutations within the AP0BEC3B motif TCW/WGA that are at MC2 sites; mutations within the AP0BEC3G motif TCGA/TCGA that are G to T; T to C mutations within the ADAR motif AAC/GTT that are at MC2 sites; mutations within the ADAR motif ACA TGT that are A to C mutations; mutations within the motif CCA/TGG that are T to A; A to G mutations within the motif CGA/TCG that are at MCI sites; mutations within the motif CTA/TAG that are T to A. In some embodiments therefore, the methods may involve detection of targeted somatic mutation (i.e. including an assessment of both the type of mutation and the codon context of the mutation) to determine whether a SNV is attributable to a deaminase (as described in, for example, WO2014/066955 and WO2017/031551).
4.2 MC-3 sites
[0077] The methods of the present disclosure in some instances comprise detecting SNVs at the third position in the mutated codon, i.e. the MC-3 site. As noted above and elsewhere, many of the deaminases have a preference for targeting nucleotides at a particular position within the mutated codon . As such, the number and/or percentage of SNVs at a MC-3 site can be a genetic indicator of deaminase activity. In some instances, a detected aberration in this percentage is indicative of "off target" SHM, which can be associated with progressing oncogenesis.
4.3 Mutations targeting a particular nucleotide
[0078] The percentage of mutations from a particular mutated nucleotide (e.g.
A, T, C or G) can also be used as a genetic indicator of deaminase activity given that particular deaminases can have a preference for targeting a particular nucleotide in a nucleic acid molecule. For example, adenosines are often the target of ADAR, while cytosines are often the target of AID. Thus, the methods of the present disclosure can include determining the percentage of SNVs resulting from a mutation of an adenine nucleotide (i.e. detecting the total number of mutations of A to C, A to T and A to G and expressing this total as a percentage of the total number of SNVs detected) ; the percentage of SNVs resulting from a mutation of a thymine nucleotide (i.e. detecting the total number of mutations of T to C, T to A and T to G and expressing this total as a percentage of the total number of SNVs detected) ; the percentage of SNVs resulting from a mutation of a cytosine nucleotide (i.e. detecting the total number of mutations of C to A, C to T and C to G and expressing this total as a percentage of the total number of SNVs detected) ; and/or the percentage of SNVs resulting from a mutation of a guanine nucleotide (i.e. detecting the total number of mutations of G to C, G to T and G to A and expressing this total as a percentage of the total number of SNVs detected) .
4.4 Strand bias
[0079] Evidence of strand bias of mutations can also be an indicator of deaminase activity. Strand bias can be assessed by calculating various ratios of mutations targeting a particular nucleotide (described above). For example, the ratio of the percentage of SNVs that include mutation of a cytosine nucleotide to the percentage of SNVs that include mutation of a guanine nucleotide (C:G ratio) can be determined. In another example, the ratio of the percentage of SNVs that include mutation of an adenine nucleotide to the percentage of SNVs that include mutation of a thymine nucleotide (A:T ratio) is determined. In a further example, the ratio of the percentage of SNVs that include mutation of an adenine or a thymine nucleotide to the percentage of SNVs that include mutation of a cytosine or a guanine nucleotide (AT:GC ratio) is determined.
4.5 ADAR-associated indicators
[0080] As demonstrated herein, mutations associated with ADAR activity can be particular useful in determining the likelihood of HBV recrudescence. Non-limiting examples of genetic indicators of ADAR activity include the number or percentage of SNVs resulting from a mutation of A (i.e. A>T, A>G and A>C); and the number or percentage of SNVs that are within an ADAR motif such as one described above (e.g. the motif WA), optionally specifying the mutation type and/or the MC site, e.g. the number or percentage of total SNVs that are within the motif WA/TW; the number or percentage of total SNVs that are within the motif AAA/TTT; number or percentage of SNVs within the motif ACA/TGT that are A>C mutations; number or percentage of T>C mutations within the motif AAC/GTT that are at MC2 sites; number or percentage of SNVs within the motif CCA/TGG that are T>A; number or percentage of A>G mutations within the motif CGA/TCG that are at MCI sites; number or percentage of SNVs within the motif CTA TAG that are T>A; number or percentage of all SNVs that are transitions at the WA/TW motif; number or percentage of SNVs within the WA/TW motif that are at MCI sites; number or percentage of SNVs within the WA/TW motif that are at MC2 sites; number or percentage of SNVs within the WA/TW motif that are at MC3 sites; number or percentage of A>G mutations within the WA/TW motif that are at MCI sites; number or percentage of A>G mutations within the WA/TW motif that are at MC2 sites; number or percentage of A>G mutations within the WA/TW motif that are at MC3 sites; number or percentage of T>C mutations within the WA/TW motif that are at MCI sites; number or percentage of T>C mutations within the WA/TW motif that are at MC2 sites; number or percentage of T>C mutations within the WA/TW motif that are at MC3 sites; number or percentage of SNVs within the WA motif that are A>G mutations; number or percentage of SNVs within the WA motif that are A>C mutations; number or percentage of SNVs within the WA motif that are A>T mutations; number or percentage of SNVs within the TW motif that are T>C mutations; number or percentage of SNVs within the TW motif that are T>G mutations; number or percentage of SNVs within the TW motif that are T>A mutations; number or percentage of SNVs within the WA/TW motif that are transitions.
4.6APOBEC-associated indicators
[0081] Mutations associated with APOBEC activity, and in particular APOBEC3B or APOBEC3G activity, can also be particularly useful in determining the likelihood of HBV recrudescence.
[0082] Exemplary genetic indicators of APOBEC3B activity include the number or percentage of mutations that are within an APOBEC3B motif such as one described above, optionally specifying the mutation type and/or the MC site, e.g. the number or percentage of mutations at motifs TCA/TGA, TC/GA and/or TCW/WGA; number or percentage of all SNVs that are transitions at the TCW/WGA motif; number or percentage of SNVs within the TCW/WGA motif that are at MCI sites; number or percentage of SNVs within the TCW/WGA motif that are at MC2 sites; number or percentage of SNVs within the TCW/WGA motif that are at MC3 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MCI sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MCI sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of SNVs within the TCW motif that are C>T mutations; number or percentage of SNVs within the TCW motif that are C>A mutations; number or percentage of SNVs within the TCW motif that are C>G mutations; number or percentage of SNVs within the WGA motif that are G>A mutations; number or percentage of SNVs within the WGA motif that are G>T mutations; number or percentage of SNVs within the WGA motif that are G>C mutations; number or percentage of SNVs within the TCW/WGA motif that are transitions.
[0083] Exemplary genetic indicators of APOBEC3G activity include the number or percentage of mutations that are within an APOBEC3G motif such as one described above, optionally specifying the mutation type and/or the MC site, e.g. the number or percentage of mutations at motifs CC/GG, CG/CG, CG/CG, TCG/CGA, and/or CGG/CCGA; the number or percentage of C to T mutations at the CC motif; the number or percentage of C to T mutations at the CG motif; the number or percentage of G to A mutations at the CG motif; the number or percentage of mutations within the motif TCGA/TCGA that are G to T; number or percentage of all SNVs that are transitions at the CC/GG motif; number or percentage of SNVs within the CC/GG motif that are at MCI sites; number or percentage of SNVs within the CC/GG motif that are at MC2 sites; number or percentage of SNVs within the CC/GG motif that are at MC3 sites; number or percentage of C>T mutations within the CC/GG motif that are at MCI sites; number or percentage of C>T mutations within the CC/GG motif that are at MC2 sites; number or percentage of C>T mutations within the CC/GG motif that are at MC3 sites; number or percentage of G>A mutations within the CC/GG motif that are at MCI sites; number or percentage of G>A mutations within the CC/GG motif that are at MC2 sites; number or percentage of G>A mutations within the CC/GG motif that are at MC3 sites; number or percentage of SNVs within the CC motif that are C>T mutations; number or percentage of SNVs within the CC motif that are C>A mutations; number or percentage of SNVs within the CC motif that are C>G mutations; number or percentage of SNVs within the GG motif that are G>A mutations; number or percentage of SNVs within the GG motif that are G>T mutations; number or percentage of SNVs within the GG motif that are G>C mutations; number or percentage of SNVs within the CC/GG motif that are transitions. 4.7 Assessinq a nucleic acid molecule for genetic indicators of endogenous deaminase activity
[0084] Any method known in the art for obtaining and assessing the sequence of a nucleic acid molecule can be used in the methods of the present invention. The nucleic acid molecule analysed using the methods of the present invention can be any nucleic acid molecule, although is generally DNA (including cDNA). Typically, the nucleic acid is mammalian nucleic acid, such as human nucleic acid, and is from a subject previously diagnosed with HBV and undergoing antiviral therapy. The nucleic acid can be obtained from any biological sample. For example, the biological sample may comprise a bodily fluid, tissue or cells. In particular examples, the biological sample is a bodily fluid, such as saliva or blood. In some examples, the biological sample is a biopsy. A biological sample comprising tissue or cells may from any part of the body and may comprise any type of cells or tissue, such as, for example, cells from the liver.
[0085] The nucleic acid molecule can contain a part or all of one gene, or a part or all of two or more genes, and it is the sequence of this gene or genes that is analysed according to the methods of the invention. Most typically, the nucleic acid molecule comprises the whole genome or whole exome, and it is the sequence of the whole genome or whole exome that is analysed in the methods of the invention.
[0086] When using the methods of the present invention, the sequence of the nucleic acid molecule may have been predetermined. For example, the sequence may be stored in a database or other storage medium, and it is this sequence that is analysed according to the methods of the invention. In other instances, the sequence of the nucleic acid molecule must be first determined prior to employment of the methods of the invention. In particular examples, the nucleic acid molecule must also be first isolated from the biological sample.
[0087] In particular examples, the biological sample from which the nucleic acid is obtained is a saliva sample or a blood sample. As demonstrated herein, there is strong concordance between the data generated from an analysis of nucleic acid obtained from saliva and the data generated from an analysis of nucleic acid obtained from blood of the same subject.
[0088] Methods for obtaining nucleic acid and/or sequenci ng the nucleic acid are well known in the art, and any such method can be utilized for the methods described herein. In some instances, the methods include amplification of the isolated nucleic acid prior to sequencing, and suitable nucleic acid amplification techniques are well known to a person of ordinary skill in the art. Nucleic acid sequencing techniques are well known in the art and can be applied to single or multiple genes, or whole exomes or genomes. These techniques include, for example, capillary sequencing methods that rely upon 'Sanger sequencing' (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467) (i.e., methods that involve chain-termination sequencing), as well as "next generation sequencing" techniques that facilitate the sequencing of thousands to millions of molecules at once. Such methods include, but are not limited to, pyrosequencing, which makes use of luciferase to read out signals as individual nucleotides are added to DNA templates; "sequencing by synthesis" technology (Illumina), which uses reversible dye- terminator techniques that add a single nucleotide to the DNA template in each cycle; and SOLiD™ sequencing (Sequencing by Oligonucleotide Ligation and Detection; Life Technologies), which sequences by preferential ligation of fixed-length oligonucleotides. These next generation sequencing techniques are particularly useful for sequencing whole exomes and genomes.
[0089] Once the sequence of the nucleic acid molecule is obtained, SNVs are then identified. SNVs may be identified by comparing the sequence to a reference sequence. The reference sequence may be the sequence of a nucleic acid molecule from a database, such as reference genome. In particular examples, the reference sequence is a reference genome, such as GRCh38 (hg38), GRCh37 (hg l9), NCBI Build 36.1 (hg l8), NCBI Build 35 (hg l7) and NCBI Build 34 (hg l6) . In some embodiments, the SNVs are reviewed to remove known and/or common single nucleotide polymorphisms (SNPs) from further analysis, such as those identified in the various SNP databases that are publically available (and, for example, where a "common" SNP is one that has at least one lOOOGenomes population with a minor allele of frequency >= 1% and for which 2 or more founders contribute to that minor allele freq uency). In further embodiments, only those SNVs that are within a coding region of an ENSEMBL gene are selected for further analysis. In addition to identifying the SNVs, the codon containing the mutation and the position of the mutation within the codon (MC-1, MC-2 or MC-3) may be identified. Nucleotides in the flanking 5' and 3' codons are also identified so as to identify the motifs. Typically, for the methods of the present invention, the sequence of the non-transcribed strand (equivalent to the cDNA sequence) of the nucleic acid molecules is analysed . In some instances, the sequence of the transcribed strand is analysed.
5. Kits and Systems for Detecting SNVs
[0090] All the essential materials and reagents required for detecting SNVs and further identifying the likelihood of HBV recrudescence, and related methods as described herein, may be assembled together in a kit. For example, when the methods of the present invention include first isolating and/or sequencing the nucleic acid to be analysed, kits comprising reagents to facilitate that isolation and/or sequencing are envisioned. Such reagents can include, for example, primers for amplification of DNA, polymerase, dNTPs (including labelled dNTPs), positive and negative controls, and buffers and solutions. Such kits will also generally comprise, in suitable means, distinct containers for each individual reagent. The kit can also feature various devices, and/or printed instructions for using the kit.
[0091] In some embodiments, the methods described generally herein a re performed, at least in part, by a processing system, such as a suitably programmed computer system. A stand-alone computer, with the microprocessor executing applications software allowing the above-described methods to be performed, may be used. Alternatively, the methods can be performed, at least in part, by one or more processing systems operating as part of a distributed architecture. For example, a processing system can be used to identify mutation types, the codon context of a mutation and/or motifs within one or more nucleic acid sequences. In some examples, commands inputted to the processing system by a user assist the processing system in making these determinations.
[0092] In one example, a processing system includes at least one microprocessor, a memory, an input/output device, such as a keyboard and/or display, and an external interface, interconnected via a bus. The external interface can be utilised for connecting the processing system to peripheral devices, such as a communications network, database, or storage devices. The microprocessor can execute instructions in the form of applications software stored in the memory to allow the methods of the present invention to be performed, as well as to perform any other required processes, such as communicating with the computer systems. The applications software may include one or more software modules, and may be executed in a suitable execution environment, such as an operating system environment, or the like.
6. Diagnostic and Therapeutic Applications
[0093] Using the methods described herein to detect SNVs in the nucleic acid molecule of a subject and measure one or more genetic indicators of endogenous deaminase activity, the likelihood of HBV recrudescence in a subject can be determined. Such a determination can facilitate the prescribing of a treatment regimen for a subject in whom HBV is likely to recrudesce. For example, the determination can assist with deciding whether antiviral treatment should begin, or whether current antiviral therapy should continue, be adjusted (e.g. be increased or reduced or altered in quality) or be stopped.
[0094] The methods can be used to obtain a profile of genetic indicators of deaminase activity for a subject, i.e. a sample profile, which can then be compared to a reference profile of genetic indicators of deaminase activity. Profiles of the present disclosure reflect an evaluation of at least any 1 or more, typically 2 or more, genetic indicators of deaminase activity associated with HBV recrudescence as described above. Reference profiles correlate with a risk or likelihood of HBV recrudescence and are typically predetermined, although can also be determined at the time of or after determining a sample profile.
[0095] Reference profiles are determined based on data obtained in the evaluation of genetic indicators of deaminase activity in individuals that have a known risk or likelihood of HBV recrudescence. Thus, for example, the reference profiles can be based on the data obtained in the evaluation of genetic indicators of deaminase activity over time in individuals that have chronic HBV, were on antiviral therapy and then stopped antiviral therapy, and relapsed . Accordingly, the reference profile can correlate with, for example, a high risk of HBV recrudescence. In other examples, the reference profile is based on the data obtained in the evaluation of genetic indicators of deaminase activity over time in individuals that have chronic HBV, were on antiviral therapy and then stopped antiviral therapy, and recovered . In such instances, the reference profile correlates to, for example, no risk or a negligible or low risk of HBV recrudescence. The individuals used to generate the reference profile may be age, gender and/or ethnicity matched or not.
[0096] A profile of genetic indicators of deaminase activity comprises at least one measurement. However, in some embodiments, the profile represents an assessment of at least 2 indicators (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more). Where the profile comprises two or more indicators, the indicators can be associated with the same or different deaminases.
[0097] In some embodiments, reference profiles encompass classification models, such as those formed using machine leaning techniques. Classification models can be formed using any suitable statistical classification or learning method that attempts to segregate bodies of data into classes based on objective parameters present in the data. Classification methods may be either supervised or unsupervised . Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition : A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, the teachings of which are incorporated by reference. Non-limiting examples of techniques that can be used to produce classification models include deep learning techniques such as Deep Boltzmann Machine, Deep Belief Networks, Convolutional Neural Networks, Stacked Auto Encoders; ensemble techniques such as Random Forest, Gradient Boosting Machines, Boosting, Bootstrapped Aggregation, AdaBoost, Stacked Generalization, Gradient Boosted Regression Trees; neural network techniques such as Radial Basis Function Network, Perceptron, Back- Propagation, Hopfield Network; regularization methods such as Ridge Regression, Least Absolute Shrinkage and Selection Operator, Elastic Net, Least Angle Regression; regression methods such as Linear Regression, Ordinary Least Squares Regression, Multiple Regression, Probit Regression, Stepwise Regression, Multivariate Adaptive Regression Splines, Locally Estimated Scatterplot Smoothing, Logistic Regression, Support Vector Machines, Poisson Regression, Negative Binomial Regression, Multinomial Logistic Regression; Bayesian techniques such as Naive Bayes, Average One-Dependence Estimators, Gaussian Naive Bayes, Multinomial Naive Bayes, Bayesian Belief Network, Bayesian Network; decision trees such as Classification and Regression Tree, Iterative Dichotomiser, C4.5, C5.0, Chi-squared Automatic Interaction Detection, Decision Stump, Conditional Decision Trees, M5; dimensionality reduction such as Principle Component Analysis, Partial Least Squares Regression, Sammon Mapping, Multidimensional Scaling, Projection Pursuit, Principle Component Regression, Partial Least Squares Discriminant Analysis, Mixture Discriminant Analysis, Quadratic Discriminant Analysis, Regularized Discriminant Analysis, Flexible Discriminant Analysis, Linear Discriminant Analysis, t- Distributed Stochastic Neighbour Embedding; instance-based techniques such as K- Nearest Neighbour, Learning Vector Quantization, Self-Organizing Map, Locally Weighted Learning; clustering methods such as k-Means, k-Modes, k-Medians, DBSCAN, Expectations Maximization, Heirarchical Clustering; adaptations, extensions, and combinations of the previously mentioned approaches
[0098] Data from individuals with chronic HBV who were on antiviral therapy and then stopped antiviral therapy, and either recovered or relapsed can be used to train a classification model . Such data is typically referred to as a training data set. Once trained, the classification model can recognize patterns in data generated using unknown samples, e.g. the data from patients with chronic HBV used to generate the sample profiles. The sample profile can then be applied to the classification model to classify the sample profile into classes, e.g. likely to experience HBV recrudescence or unlikely to experience HBV recrudescence.
[0099] In other examples, HBV recrudescence will be determined to be likely when 1, 2, 3, 4, 5 or more genetic indicators of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity. In a particular example, HBV recrudescence will be determined to be likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for that genetic indicator of endogenous deaminase activity. Conversely, HBV recrudescence will be determined to be unlikely when one or more of these genetic indicators of endogenous deaminase activity are within the respective predetermined range intervals. In a particular example, HBV recrudescence will be determined to be unlikely when none of the genetic indicators of endogenous deaminase activity are within the respective predetermined range intervals (i.e. no genetic indicators of endogenous deaminase activity are outside a predetermined normal range interval for genetic indicators of endogenous deaminase).
[OIOO] In some examples, a score is attributed to each genetic indicator of endogenous deaminase activity that is outside a predetermined range interval, and the total score is then calculated by adding all of the scores. HBV recrudescence will then be determined to be likely when the score is equal to or above a threshold score. The score for each genetic indicator of endogenous deaminase activity may be the same or may be different (e.g. may be "weighted" such that one genetic indicator of endogenous deaminase activity that is outside a predetermined range interval might be given a score that is more than another genetic indicator of endogenous deaminase activity that is outside a predetermined range interval). In a particular example, each genetic indicator of endogenous deaminase activity that is outside a predetermined range interval is given a score of 1. In some embodiments, the threshold score is 1 such that any score of 1 and above results in a determination that HBV recrudescence is likely.
[0101] The predetermined normal range interval for a genetic indicator of endogenous deaminase activity can be determined by assessing a genetic indicator of deaminase activity in two or more non-diseased or healthy subjects (e.g. subjects that do not have HBV infection and/or who do not have any other known infection or disease). A normal range interval for the genetic indicator is then calculated to set the upper and lower limits of what would be considered normal values for that indicator, e.g. values that would reflect a normal deaminase activity in healthy individuals. In a particular example, the normal range interval is calculated by measuring the average plus or minus 2 standard deviations, whereby the lower limit of the range interval is the average minus 2 standard deviations and the upper limit of the range interval is the average plus 2 standard deviations. In other examples, less than or more than 2 standard deviations are used to set the upper and lower limits of the interval, such as 1, 1.5, 2.5, 3, 3.5 or more standard deviations. In still further examples, the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves. In still further examples, the upper and lower limits of the predetermined normal range interval are established using receiver operating characteristic (ROC) curves. The subjects used to determine the predetermined normal range interval can be of any age, sex or background, or may be of a particular age, sex, ethnic background or other subpopulation. Thus, in some embodiments, two or more predetermined normal range intervals can be calculated for the same genetic indicator of deaminase activity, whereby each range interval is specific for a particular subpopulation, e.g. a particular sex, age group, ethnic background and/or other subpopulation.
[0102] In some embodiments, the subject has chronic HBV infection, and the methods of the present disclosure are performed to determine the likelihood of HBV recrudescence in these subjects. In a particular embodiment, the subject has chronic HBV and is on antiviral therapy, and the methods of the present disclosure are performed to determine the likelihood of HBV recrudescence in these subjects if they terminate antiviral therapy. In alternative embodiments, the subject does not have chronic HBV infection. For example, the subject may have experienced or may be experiencing an acute HBV infection, and may appear to be resolving or to have resolved that infection (with or without antiviral therapy). The methods of the present disclosure can therefore be performed to determine the likelihood of HBV recrudescence in these subjects.
[0103] As well as being of use to determine the likelihood of HBV recrudescence, an analysis of genetic indicators of deaminase activity as described herein can also be used to assist in determining whether or not antiviral therapy is working, and/or in monitoring disease progression . For example, nucleic acid obtained from biological samples from a subject over time (e.g. over time during therapy; before therapy has commenced and/or after therapy has commenced) can be assessed as described above to measure one or more genetic indicators of endogenous deaminase activity. If there is an increase over time (i.e. from a first sample to a second sample) in the number of indicators that are outside the predetermined normal range interval, or an increase over time in a value of one or more indicators, then it can be determined that the therapy is likely to be ineffective and/or the disease is likely to have progressed. Conversely, if there is a decrease or no change over time in the number of indicators that are outside the predetermined normal range interval, or an increase or no change over time in a value of one or more indicators, then it can be determined that it is likely that the therapy is effective and/or the disease has stabilised, not progressed and/or regressed.
[0104] The methods of the present invention also extend to therapeutic or preventative protocols. In instances where HBV recrudescence is determined to be unlikely, or disease regression is determined to be likely, existing antiviral protocols may be amended to reduce the antiviral therapy (e.g. reduce dosage), or to remove a subject from a therapy completely. In instances where HBV recrudescence or disease progression is determined to be likely, protocols designed to reduce that likelihood may be designed and applied to a subject. For example, when the subject is already on antiviral therapy, that therapy can be continued or altered (e.g . by increasing the dosage and/or altering the type of therapy. In instances where the subject is not yet on an antiviral therapy, an appropriate antiviral therapy can be designed for the subject and administered. The therapeutic methods can involve obtaining a biological sample from the subject (e.g . a blood or saliva sample), performing the methods described herein to determine whether or not HBV recrudescence is likely, and then exposing the subject to, or administering to the subject, antiviral therapy if it is determined that HBV recrudescence is likely. Alternatively, the therapeutic methods can involve sending a biological sample obtained from a subject to a laboratory to (i) conduct the methods of described herein to determine whether or not HBV recrudescence is likely, and (ii) provide the results of the method, wherein the results comprise a determination of whether HBV recrudescence is likely or unlikely, receiving the results from step (a); and then exposing the subject to, or administering to the subject, an antiviral therapy if the results comprise a determination that HBV recrudescence is likely.
[0105] Antiviral therapies appropriate for the treatment of HBV are well known in the art and include, for example, immunotherapy (e.g. interferon therapy, including pegylated IFN-a therapy), or nucleotide/nucleoside therapy (e.g. lamivudine (e.g. Epivir- HBV®), emtricitabine (e.g. Emtriva®) fovir dipivoxil (e.g. Hepsera®), entecavir (e.g. Baraclude®), telbivudine (e.g . Tyzeka®), clevudine (e.g. Levovir®) and tenofovir disoproxil (e.g. Viread®) .
[0106] The present invention can be practiced in the field of predictive medicine for the purposes of predicting, detection or monitoring the recrudescence of HBV in a subject, and/or monitoring disease progression or response to therapy efficacy.
[0107] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES
Example 1
Materials and Methods
Sample collection and DNA preparation
[0108] Saliva and/or blood samples were obtained from subjects enrolled in a clinical trial at Monash Medical Centre, Victoria. The subjects enrolled in the trial included healthy subjects and subjects with chronic HBV infection and on therapy. Subjects with chronic HBV infection were HBeAg negative and on nucleoside/nucleotide (NA) therapy (tenofovir, entecavir, adefovir or lamivudine), and met current APASL guidelines for consideration of antiviral cessation (uninterrupted nucleoside treatment for > 2years and undetectable serum HBV DNA on three separate occasions > 6 months apart (undetectable defined by a value < lower limit of detection using a sensitive commercial PCR assay). The chronic HBV subjects also had normal serum alanine aminotransferase (ALT) levels and minimal to moderate liver fibrosis (defined as METAVIR liver fibrosis stage FO - F3 inclusive prior to initial NA therapy and/or transient liver elastogram (TLE) (Fibroscan®) <9.6 kPa at screening). The samples were taken from the chronic HBV subjects just prior to termination of NA therapy.
[0109] All saliva samples were collected using the Oragene DNA (OG-500) saliva collection kit. The DNA was extracted from the OG-500 saliva samples by Smart DNA located at the Monash Clinical Trial Precinct (MCTP) using QIAsymphony DSP midi kit CAT NO: 937255.
[0110] Blood samples were collected and DNA extracted from the samples using standard techniques.
Exome library preparation and sequencing
[0111] Exome libraries were made using Agilent SureSelectXT Target
Enrichment System according to protocol G7530- 90000 Version B5 June 2016. Capture Probes: Agilent SureSelect Clinical Research Exome Cat No 5190-7344; Design ID S06588914. All libraries were verified by Qubit, bioanalyzer and qPCR.
[0112] Exome sequencing was performed on a HiSeq3000 with 100 base pair, paired end sequencing and four samples per lane. This provided between 84 and 104 million reads per sample. On average, coverage was greater than lOOx. Reads were analysed with FastQC for quality
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) . Adaptors were trimmed with cutadapt and genomic mapping to hg37 was performed with BWA (Li H and Durbin R Bioinformatics (2009) 25: 1754-60.). LiftOver (http://genome.ucsc.edu/) was used to transform from hg37 to hg38 genomic coordinates. PCR duplicates were marked with picard (http://broadinstitute.github.io/picard). Single nucleotide variants (SNVs) that had quality scores of 100 or more and that were not in SNPdb were selected for further analysis.
Detection of mutations attributable to deaminases
[0113] SNVs with quality scores of over 100 which were not in SNPdb (as described above), and which were within a coding reg ion of an ENSEMBL gene were then further analysed using the following steps:
a) the codon context within the structure of the mutated codon (MC) was determined, i.e. the position of the SNV within the encoding triplet was determined, wherein the first position (read from 5' to 3') is referred to as MCI, the second position is referred to as MC2 and the third position is referred to as MC3;
b) a nine-base window was extracted from the surrounding genome sequence such that the sequence of three complete codons was obtained. The direction of the gene was used for determining 5' and 3' directions, and for determining the correct strand of the nine bases. The nine-base window was always reported according to the direction of the gene such that bases in the window around variants in genes on the reverse strand of the genome are reverse complimented in relation to the genome, but in the forward direction in relation to the gene. By convention, this context is always reported in the same strand of the gene. Positive strand genes will have codon context bases from the positive strand of the reference genome, and negative strand genes will have codon context bases from the negative strand of the reference genome;
c) motif searching was performed using known motifs for the fou r main deaminases to determine whether the variation was within such a motif and thus resulted from deaminase activity, with the four main deaminase motifs being as follows (wherein the underlined base corresponds to the targeted/mutated base, and the target motif to the right of the forward slash is the reverse compliment of the forward strand motif that is used for searching on the reverse strand of the gene) :
AID - WRC/GYW
ADAR - WA/TW
APOBEC3G - CC/GG
APOBEC3B - TCA/TGA On this basis, SNVs attributable to AID, ADAR, APOBEC3G or APOBEC3B were identified. The number of SNVs at particular positions within the mutated codon were also calculated, as were the number of SNVs that involved mutation of an A, T, C or G to another nucleotide.
Example 2
Correlation between data obtained using blood and saliva samples
[0114] To determine whether saliva samples could be effectively used in methods for detecting mutations attributable to deaminases, saliva and blood samples from five healthy subjects were collected and analysed as described in Example 1. The samples included multiple saliva samples taken from each single subject in the same day, with one blood and three saliva samples taken from each of the five subjects. Reference intervals for saliva samples were calculated as the mean +/- 2 standard deviations (2STDEV) .
[0115] As shown in Table 2, below, there is strong concordance between the number of mutations attributable to each of the assessed deaminases (i.e., AID, ADAR, APOBEC3G and APOBEC3B) as measured in the saliva or blood, and as measured between each saliva sample from the same subject. All results using the saliva samples fell within the reference intervals, while 13 of 15 results using the blood samples fell within the reference interval (the calculated number of mutations attributable to ADAR activity in patient 1 and the calculated number of mutations attributable to AID activity were slightly above the reference intervals). These results indicate that saliva samples can be used to accurately assess mutations attributable to deaminase activity in a subject.
Table 2. Mutations in nucleic acid obtained from saliva and blood of healthy subjects attributable to deaminases
Figure imgf000036_0001
Patient ID: HP_2 (total SNVs: 319)
Sample type Saliva Saliva Saliva Blood Saliva Saliva
Sample ID 2_1 2_2A 2_2 1_3 AVERAGE 2STDEV
AID mutations 12 15 12 12 13.0 3.5
ADAR mutations 23 25 30 27 26.0 7.2
A3G mutations 60 50 61 60 57.0 12.2
A3B mutations 13 16 11 15 13.3 5.0
Patient ID: HP_3 (total SNVs: 300)
Sample type Saliva Saliva Saliva Blood Saliva Saliva
Sample ID 3_1 3_2A 3_2 3_3 AVERAGE 2STDEV
AID mutations 11 14 12 14 12.3 3.1
ADAR mutations 24 27 25 26 25.3 3.1
A3G mutations 49 51 58 52 52.7 9.5
A3B mutations 14 12 16 13 14.0 4.0
Patient ID: HP_4 (total SNVs: 277)
Sample type Saliva Saliva Saliva Blood Saliva Saliva
Sample ID 4_1 4_2A 4_2 4_3 AVERAGE 2STDEV
AID mutations 10 15 13 13 12.7 5.0
ADAR mutations 20 29 25 24 24.7 9.0
A3G mutations 56 68 69 60 64.3 14.5
A3B mutations 9 13 12 14 11.3 4.2
Patient ID: HP_5 (total SNVs: 314)
Sample type Saliva Saliva Saliva Blood Saliva Saliva
Sample ID 5_1 5_2A 5_2 5_3 AVERAGE 2STDEV
AID mutations 7 8 8 9 7.7 1.2
ADAR mutations 23 26 21 28 23.3 5.0 A3G mutations 74 79 77 77 76.7 5.0
A3B mutations 17 18 14 16 16.3 4.2
Example 3
Determination of baseline deaminase activity in healthy subjects
[0116] The SNVs in 24 healthy subjects (all Caucasian) were assessed to identify the number of SNVs of various types in healthy individuals, so as to determine the total mutation burden (i.e. the total number of SNVs) and then calculate normal range intervals for various genetic indicators of deaminase activity. Specifically, these indicators included the percentage of SNVs at the targeted nucleotide of the AID motif; the percentage of SNVs at the targeted nucleotide of the ADAR motif; the percentage of SNVs at the targeted nucleotide of the APOBEC3G motif; the percentage of SNVs at the targeted nucleotide of the APOBEC3B motif; the percentage of SNVs at an MC-3 site; the percentage of SNVs that were mutations of an adenine; the percentage of SNVs that were mutations of a thymine; and the percentage of SNVs that were mutations of a cytosine. To detect strand bias, the ratio of the percentage of SNVs that were mutations of a cytosine to the percentage of SNVs that were mutations of a guanine (C:G ratio); the ratio of the percentage of SNVs that were mutations of an adenine to the percentage of SNVs that were mutations of a thymine (A:T ratio) ; and the ratio of the percentage of SNVs that were mutations of an adenine or a thymine to the percentage of SNVs that were mutations of a guanine or cytosine (AT:GC ratio) were calculated. The average and standard deviations for each of these values was determined, and the range interval calculated as the average +/- 2 standard deviations.
[0117] Table 3 provides the results of the study, and sets forth the normal range intervals for each of the genetic indicators of deaminase activity.
TABLE 3
Patient Mutation AID ADAR APOBEC APOBEC All MC3 All off A's All off All off C's STRAND - BIAS
3G 3B T's
Sample ID Burden % % % % % % % % C:G A:T AT:GC
H P_1_1 332 4.82 6.33 19.28 6.93 36.75 18.07 14.46 38.55 1.33 1.18 0.48
H P_2_1 319 3.76 7.21 18.81 4.08 34.80 20.69 16.61 37.93 1.53 1.25 0.60
H P_3_1 300 3.67 8.00 16.33 4.67 37.00 23.33 16.67 30.67 1.05 1.40 0.67
H P_4_1 277 3.61 7.22 20.22 3.25 35.74 20.58 17.69 34.66 1.28 1.16 0.62
H P_5_1 314 2.23 7.32 23.57 5.41 36.94 21.34 15.29 41.72 1.93 1.40 0.58
H P_6_1 312 4.81 5.77 24.68 4.81 36.54 16.67 15.06 37.50 1.22 1.11 0.46
H P_7_1 340 3.24 8.53 17.94 4.12 39.41 20.59 16.76 35.00 1.27 1.23 0.60
H P_8_1 363 3.03 7.99 21.21 4.68 38.02 19.01 19.56 34.44 1.28 0.97 0.63
H P_9_1 291 3.78 6.87 14.78 4.81 30.93 20.62 17.87 34.36 1.27 1.15 0.63
HP_10_1 366 3.28 8.47 22.13 5.74 34.70 20.22 15.30 33.88 1.11 1.32 0.55
HP_11_1 384 3.91 8.33 19.79 5.21 35.68 20.57 15.89 35.16 1.24 1.30 0.57
HP_12_1 321 4.05 8.72 17.45 5.30 37.38 20.56 18.07 36.14 1.43 1.14 0.63
HP_13_1 327 3.06 6.73 19.88 5.20 36.09 21.71 15.29 37.31 1.45 1.42 0.59
HP_14_1 291 3.44 7.90 19.59 3.78 33.68 23.37 13.40 35.40 1.27 1.74 0.58
HP_15_1 368 5.43 9.24 20.92 5.43 33.70 21.74 14.67 34.51 1.19 1.48 0.57
HP_16_1 293 3.41 8.53 17.06 5.12 38.57 22.53 19.45 30.72 1.13 1.16 0.72
HP_17_1 364 2.47 8.24 18.68 4.95 39.56 19.78 17.58 29.12 0.87 1.13 0.60
HP_18_1 306 5.88 8.17 19.28 2.29 39.87 18.30 18.30 36.93 1.40 1.00 0.58
HP_19_1 332 1.81 7.23 18.37 5.72 37.05 20.48 17.47 36.45 1.42 1.17 0.61
HP_20_1 375 3.73 5.60 22.67 4.53 36.00 17.87 13.33 37.07 1.17 1.34 0.45
HP_21_1 313 4.15 6.71 21.09 5.75 34.82 18.85 18.21 36.74 1.40 1.04 0.59
HP_23_1 304 4.28 7.57 19.08 5.59 39.14 19.08 18.42 33.55 1.16 1.04 0.60
HP_24_1 320 4.06 9.38 16.88 5.63 40.94 24.06 15.94 34.38 1.34 1.51 0.67
RANGE 277.00 1.80 5.60 14.78 2.29 30.93 16.67 13.33 29.12 0.87 0.97 0.44
INTERVALS 384.00 5.88 9.38 23.57 6.93 40.94 24.06 19.56 41.72 1.93 1.74 0.72
Example 4
Mutation profiles of subjects with chronic HBV
[0118] The nucleic acid from subjects with chronic HBV just prior to termination of NA therapy was assessed as described above in Examples 1 and 3. The clinical markers of HBV recrudescence (e.g. serum HBV DNA levels and alanine aminotransferase (ALT) levels) were also assessed in these subjects subsequent to termination of therapy to determine which subjects experienced HBV recrudescence (or flare of disease) .
[0119] As can be seen in Table 4, in all chronic HBV subjects that experienced
HBV recrudescence, at least one genetic indicator of deaminase activity was outside the normal range interval (H - high or L - low compared to the normal range interval), suggesting that the subject had an impaired deaminase activity and an impaired deaminase-associated immune response. Of the chronic HBV subjects that successfully terminated NA therapy and cleared the infection, none had any genetic indicators of deaminase activity outside the normal range interval.
[0120] In particular, an aberrant percentage of SNVs at an ADAR motif was associated with HBV recrudescence. This suggested that dysregulation of ADAR activity (e.g. editing) may be associated with HBV recrudescence, a result consistent with the teachings in the prior art. These results indicate that an aberrant percentage of SNVs at an ADAR motif is an effective predictor of HBV recrudescence. Other genetic indicators that appeared predictive of HBV recrudescence included an aberrant percentage of SNVs at an MC-3 site (possibly an indicator of 'off-target' somatic hypermutation (SHM) which is associated with progression to oncogenesis), and an aberrant percentage of mutations of an adenine (possibly indicative of reduced ADAR or Pol-eta function and/or disrupted MMR).
[0121] A predicted test score was calculated wherein if any of the genetic indicators of deaminase activity were abnormal, a score of 1 was assigned, with the total then calculated. All subjects that cleared the infection had a test score of 0, while all subjects that experienced reactivation of HBV had a score of > 1. This results clearly demonstrate that the genetic indicators of deaminase activity can be used to predict the likelihood of HBV recrudescence in a subject should that subject terminate antiviral therapy.
[0122] It was also interesting to note that the overall mutation burden was significantly higher in all HBV patients compared to healthy subjects. This difference is largely attributed to the well-known role of APOBEC3G as a potent antiviral deaminase (noting that the overall mutation burden associated with the APOBEC3G in HBV subjects was higher than healthy subjects - data not shown). This suggests that 'off-target' SHM- like antiviral activity predominantly involving APOBEC3G has fundamentally altered the genomic DNA of chronic HBV patients compared to healthy subjects. This is consistent with the known role of APOBEC3G as a potent antiviral factor agent.
Example 5
Additional analysis of patients with chronic HBV
[0123] Additional analysis of genetic indicators of deaminase activity in whole exomes of patients enrolled in two different studies was next performed. The patients all had chronic HBV and were previously on antiviral therapy. Twelve chronic HBV patients that ceased antiviral treatment and recovered (i.e. successfully stopped treatment and did not have to restart antiviral therapy, although some 'flared' prior to recovering) were from a clinical Chronic HBV Stop Study at St Vincent's Hospital in Melbourne, Australia (patient codes starting with HBV_2 in Table 5, below). The remaining patients in this group that successfully ceased treatment, and all of the chronic HBV patients in the group that did not successfully stop treatment and had to resume antiviral therapy, were enrolled in a similar Chronic HBV Stop Study led by Professor Georgios Papatheodoridis of the Medical School of Athens. DNA was extracted from peripheral blood mononuclear ceils and sequenced and analysed as essentially described in Example 1, above. The specific genetic indicators of deaminase activity analysed included the following, and are represented in the columns of Table 5 from left to right:
• percentage of C>T mutations within the AID motif WRC/GYW that are at MCI sites
• percentage of G>A mutations within the AID motif WRC/GYW that are at MC2 sites
• percentage of C>T mutations within the motif ACA/TGT that are at MC2 sites
• percentage of G>A mutations within the motif CCA/TGG that are at MCI sites
• percentage of G>A mutations within the motif GCT/AGC that are at MC2 sites
• percentage of mutations within the APOBEC3B (A3B) motif TCW/WGA that are at MC2 sites
• percentage of mutations within the APOBEC3G (A3G) motif TCGA/TCGA that are G>T
• percentage of T>C mutations within the ADAR motif AAC/GTT that are at MC2 sites
• percentage of total mutations that are within the ADAR motif AAA TTT
• percentage of mutations within the ADAR motif ACA/TGT that are A>C mutations
• percentage of mutations within the motif CCA/TGG that are T>A
• percentage of A>G mutations within the motif CGA/TCG that are at MCI sites • percentage of mutations within the motif CT A/TAG that are T>A
[0124] The range intervals for each metric was calculated using the successfully treated/recovered patient set. As shown in Table 5, in the combined data sets from 2 different cHBV Stop Study trial sites, which consisted of a total of 60 patients, there were no false positives or false negatives. AH of the patients needing to resume antiviral therapy had at least one outlier indicating an 'out of range' measure of one or more of the key metrics selected.
[0125] Interestingly, no patient had a value outside the range interval for a indicators of deaminase activity associated with AID. This is may be because AID is an initiator of SHM-like activity involving deamination in response to a host viral infection. In contrast, the main difference between those patients that recovered and those that needed to resume treatment was seen in indicators associated with APOBEC3B, APOBEC3G and ADAR activity. Five of the twelve patients that required a resumption in treatment had values outside of the calculated range intervals for indicators of activity of APOBEC3G and APOBEC3B C-to-U deaminases, deaminases that are known to mutate viral DNA during replication. Ten of the twelve patients that required a resumption in antiviral treatment had values outside of the calculated range intervals for indicators of ADAR activity, implying disrupted A-to-I editing.
[0126] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[0127] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
[0128] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims. TABLE 4
cHBV TEST RESULTS
STRAND-BIAS [5]
Patient Actual Predicted Mutation AID ADAR A3G A3B All MC3 All off A's All off T's All off C's
Sample ID Phenotype Phenotype Burden % % % % % % % % C:G A:T AT:GC
HBV_2_2 Flare Flare 546.00 H 4.40 6.41 20.70 4.58 42.49 H 22.16 14.10 30.40 1.10 1.57 0.57
HBV_2_1 Flare Flare 521.00 H 3.84 5.57 L 19.96 3.65 36.66 17.66 13.44 31.29 1.20 1.31 0.45
HBV_2_8 Flare Flare 561.00 H 3.74 5.35 L 23.71 H 4.46 38.86 15.69 L 15.33 30.66 1.25 1.02 0.45
HBV 2 6 Mild Flare Flare 376.00 4.52 10.37 H 16.76 5.32 39.36 22.07 15.16 30.85 0.97 1.46 0.59
HBV_2_3 Clear Clear 607.00 H 4.12 7.41 19.77 4.94 37.40 20.76 15.16 32.29 1.02 1.37 0.56
HBV_2_9 Clear Clear 527.00 H 5.12 5.69 19.54 4.55 36.81 16.70 14.04 35.29 1.04 1.19 0.44
HBV_2_4 Clear Clear 525.00 H 4.57 6.10 21.71 4.38 39.05 16.95 13.52 36.38 1.10 1.25 0.44
HBV_2_5 Clear Clear 558.00 H 4.66 5.91 18.10 4.48 36.92 19.89 15.05 33.69 1.07 1.32 0.54
HBV_2_7 Clear Clear 535.00 H 4.86 6.17 19.07 3.93 34.58 17.57 15.70 34.39 1.06 1.12 0.50
HBV_2_10_ Clear Clear 522.00 H 3.45 9.20 22.03 3.26 37.36 18.39 16.86 33.52 1.07 1.09 0.54
RANGE 277.00 1.80 5.60 14.78 2.29 30.93 16.67 13.33 29.12 0.87 0.97 0.44
INTERVAL 384.00 5.88 9.38 23.57 6.93 40.94 24.06 19.56 41.72 1.93 1.74 0.72
TABLE 5
Figure imgf000044_0001
TABLE 5 CONT'D
Figure imgf000045_0001

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject, the method comprising :
analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs);
measuring one or more genetic indicators of endogenous deaminase activity based on the number and/or type of SNVs detected so as to obtain a sample profile of genetic indicators of endogenous deaminase activity; and
determining the likelihood of HBV recrudescence based on a comparison between the sample profile and a reference profile of genetic indicators of endogenous deaminase activity.
2. The method of claim 1, comprising determining that HBV recrudescence is likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for genetic indicators of endogenous deaminase activity; or determining that HBV recrudescence is not likely when no genetic indicators of endogenous deaminase activity are outside a predetermined normal range interval for genetic indicators of endogenous deaminase activity.
3. The method of claim 1, comprising assigning a score to each genetic indicator of endogenous deaminase activity that is outside a predetermined normal range interval for the genetic indicator of endogenous deaminase activity and adding each score to calculate a total score; and
determining that HBV recrudescence is likely when the total score is equal to or above a threshold score; or
determining that HBV recrudescence is not likely when the total score is less than a threshold score.
4. A method for determining the likelihood of hepatitis B virus (HBV) recrudescence in a subject, the method comprising :
analysing the sequence of a nucleic acid molecule from a subject to detect single nucleotide variations (SNVs);
measuring one or more genetic indicators of endogenous deaminase activity based on the number and/or type of SNVs detected; and determining that HBV recrudescence is likely when at least one genetic indicator of endogenous deaminase activity is outside a predetermined normal range interval for genetic indicators of endogenous deaminase activity; or
determining that HBV recrudescence is not likely when no genetic indicators of endogenous deaminase activity are outside a predetermined normal range interval for genetic indicators of endogenous deaminase activity.
5. A method for determining the likelihood of HBV recrudescence in a subject, the method comprising :
analysing the sequence of a nucleic acid molecule from a subject to detect SNVs; measuring one or more genetic indicators of endogenous deaminase activity based on the number and/or type of SNVs detected;
assigning a score to each genetic indicator of endogenous deaminase activity that is outside a predetermined normal range interval for the genetic indicator of endogenous deaminase activity and adding each score to calculate a total score; and
determining that HBV recrudescence is likely when the total score is equal to or above a threshold score; or
determining that HBV recrudescence is not likely when the total score is less than a threshold score.
6. The method of any one of claims 1 to 5, wherein the one or more genetic indicators of endogenous deaminase activity is selected from among the number or percentage of single nucleotide variations (SNVs) that are at a deaminase motif (optionally also specifying the type of mutation and/or codon context) ; the number or percentage of SNVs at MC-3 sites; the number or percentage of SNVs resulting from mutation of an adenine nucleotide; the number or percentage of SNVs resulting from mutation of a thymine nucleotide; the number or percentage of SNVs resulting from a mutation of a cytosine nucleotide; the number or percentage of SNVs resulting from mutation of a guanine nucleotide; the ratio of the percentage of SNVs resulting from mutation of a cytosine nucleotide to the percentage of SNVs resulting from a mutation of guanine nucleotide (C:G ratio); the ratio of the percentage of SNVs resulting from mutation of a adenine nucleotide to the percentage of SNVs resulting from a mutation of a thymine nucleotide (A:T ratio); the ratio of the percentage of SNVs resulting from a mutation of an adenine or a thymine nucleotide to the percentage of SNVs resulting from a mutation of a cytosine or a guanine nucleotide (AT:GC ratio).
7. The method of any one of claims 1 to 6, wherein the endogenous deaminase is selected from among activation-induced cytosine deaminase (AID), apoiipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 1 cytosine deaminase (APOBECl), APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H and an adenosine deaminase acting on RNA (ADAR).
8. The method of any one of claims 1 to 7, wherein the one or more genetic indicators of endogenous deaminase activity include the number or percentage of SNVs at an ADAR motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an APOBEC3G motif (optionally also specifying the type of mutation and/or codon context), the number or percentage of SNVs at an
APOBEC3B site (optionally also specifying the type of mutation and/or codon context); the number or percentage of the percentage of SNVs at MC-3 sites; and/or the number or percentage of SNVs resulting from mutation of an adenine nucleotide.
9. The method of claim 6 or 7, wherein the deaminase motif is an AID motif selected from among motifs comprising the nucleic acid sequence WRC/GYW and
WRCG/CGYW (wherein the underlined nucleotide is mutated).
10. The method of any one of claims 6 to 8, wherein the deaminase motif is an ADAR motif selected from among motifs comprising the nucleic acid sequence WA/TW; WAY/RTW; WTAW/WTAW; RAWA/TWTY; AAC/GTT; AAA/TXT; AC /TGT; CCA/IGG;
CG /TCG; and CTA TAG (wherein the underlined nucleotide is mutated).
11. The method of any one of claims 6 to 8, wherein the deaminase motif is an APOBEC3G motif selected from among motifs comprising the nucleic acid sequence CC/GG, CG/CG, CG/CG, TCG/CGA, CCG/CGG and CGG/CCG (wherein the underlined nucleotide is mutated).
12. The method of claim 6 to 8, wherein the deaminase motif is an APOBEC3B motif selected from among motifs comprising the nucleic acid sequence TCA/TGA, TC/GA and TCW/WGA (wherein the underlined nucleotide is mutated).
13. The method of claim 6 or 7, wherein the deaminase motif is an APOBEC3H motif comprising the nucleic acid sequence TCW/WGA (wherein the underlined nucleotide is mutated) .
14. The method of claim 6 or 7, wherein the deaminase motif is an APOBECl motif selected from among motifs comprising the nucleic acid sequence TG/CA and GG/CC (wherein the underlined nucleotide is mutated) .
15. The method of claim 6 or 7, wherein the deaminase motif is an APOBEC3A or APOBEC3F motif selected from among motifs comprising the nucleic acid sequence TC/GA and TCW/WGA (wherein the underlined nucleotide is mutated) .
16. The method of any one of claims 1 to 7, wherein the one or more genetic indicators of deaminase activity comprises a genetic indicator of ADAR activity, a genetic indicator of APOBEC3B activity and/or a genetic indicator of APOBEC3G activity.
17. The method of claim 16, wherein the genetic indicator of ADAR activity is selected from among the number or percentage of SNVs resulting from a mutation of A and the number or percentage of SNVs that are within an ADAR motif.
18. The method of claim 17, wherein the genetic indicator of ADAR activity is selected from among the number or percentage of total SNVs that are within the motif WA/TW; number or percentage of total SNVs that are within the motif AAA TTT; number or percentage of SNVs within the motif ACA XGT that are A>C mutations; number or percentage of T>C mutations within the motif AAC/GTT that are at MC2 sites; number or percentage of SNVs within the motif CCA/TGG that are T>A; number or percentage of A>G mutations within the motif CGA/TCG that are at MCI sites; number or percentage of SNVs within the motif CTA./TAG that are T>A; number or percentage of all SNVs that are transitions at the WA/TW motif; number or percentage of SNVs within the WA/TW motif that are at MCI sites; number or percentage of SNVs within the WA/TW motif that are at MC2 sites; number or percentage of SNVs within the WA/TW motif that are at MC3 sites; number or percentage of A>G mutations within the WA/TW motif that are at MCI sites; number or percentage of A>G mutations within the WA/TW motif that are at MC2 sites; number or percentage of A>G mutations within the WA/TW motif that are at MC3 sites; number or percentage of T>C mutations within the WA/TW motif that are at MCI sites; number or percentage of T>C mutations within the WA/TW motif that are at MC2 sites; number or percentage of T>C mutations within the WA/TW motif that are at MC3 sites; number or percentage of SNVs within the WA motif that are A>G mutations; number or percentage of SNVs within the WA motif that are A>C mutations; number or percentage of SNVs within the WA motif that are A>T mutations; number or percentage of SNVs within the TW motif that are T>C mutations; number or percentage of SNVs within the TW motif that are T>G mutations; number or percentage of SNVs within the TW motif that are T>A mutations; number or percentage of SNVs within the WA/TW motif that are transitions.
19. The method of claim 16, wherein the genetic indicator of APOBEC3B activity is the number or percentage of SNVs that are within an APOBEC3B motif.
20. The method of claim 19, wherein the genetic indicator of APOBEC3B activity is selected from among the number or percentage of mutations at motifs TCA/TGA, TC/GA and/or TCW/WGA; number or percentage of all SNVs that are transitions at the
TCW/WGA motif; number or percentage of SNVs within the TCW/WGA motif that are at MCI sites; number or percentage of SNVs within the TCW/WGA motif that are at MC2 sites; number or percentage of SNVs within the TCW/WGA motif that are at MC3 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MCI sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of C>T mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MCI sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC2 sites; number or percentage of G>A mutations within the TCW/WGA motif that are at MC3 sites; number or percentage of SNVs within the TCW motif that are C>T mutations; number or percentage of SNVs within the TCW motif that are C>A mutations; number or percentage of SNVs within the TCW motif that are C>G mutations; number or percentage of SNVs within the WGA motif that are G>A mutations; number or percentage of SNVs within the WGA motif that are G>T mutations; number or percentage of SNVs within the WGA motif that are G>C mutations; number or percentage of SNVs within the TCW/WGA motif that are transitions.
21. The method of claim 16, wherein the genetic indicator of APOBEC3G activity is the number or percentage of SNVs that are within an APOBEC3G motif.
22. The method of claim 21, wherein the genetic indicator of APOBEC3G activity is selected from among the number or percentage of mutations at motifs CC/GG, CG/CG,
CG/CG, TCG/CGA, and/or CGG/CCGA; the number or percentage of C to T mutations at the CC motif; the number or percentage of C to T mutations at the CG motif; the number or percentage of G to A mutations at the CG motif; the number or percentage of mutations within the motif TCGA/TCGA that are G to T; number or percentage of all SNVs that are transitions at the CC/GG motif; number or percentage of SNVs within the CC/GG motif that are at MCI sites; number or percentage of SNVs within the CC/GG motif that are at MC2 sites; number or percentage of SNVs within the CC/GG motif that are at MC3 sites; number or percentage of C>T mutations within the CC/GG motif that are at MCI sites; number or percentage of C>T mutations within the CCJGG motif that are at MC2 sites; number or percentage of C>T mutations within the CC/GG motif that are at MC3 sites; number or percentage of G>A mutations within the CC/GG motif that are at MCI sites; number or percentage of G>A mutations within the CC/GG motif that are at MC2 sites; number or percentage of G>A mutations within the CC/GG motif that are at MC3 sites; number or percentage of SNVs within the CC motif that are C>T mutations; number or percentage of SNVs within the CC motif that are OA mutations; number or percentage of SNVs within the CC motif that are C>G mutations; number or percentage of SNVs within the GG motif that are G>A mutations; number or percentage of SNVs within the GG motif that are G>T mutations; number or percentage of SNVs within the GG motif that are G>C mutations; number or percentage of SNVs within the CC/GG motif that are transitions.
23. The method of any one of claims 1 to 20, wherein the SNVs do not include known single nucleotide polymorphisms.
24. The method of any one of claims 1 to 21, wherein the nucleic acid molecule has been obtained from a blood or saliva sample from the subject.
25. The method of any one of claims 1 to 24, further comprising obtaining a biological sample from the subject and extracting the nucleic acid molecule.
26. The method of claim 25, wherein the biological sample is blood or saliva.
27. The method of any one of claims 1 to 26, wherein the subject has chronic HBV infection.
28. The method of any one of claims 1 to 27, wherein the subject is on antiviral therapy.
29. The method of claim 28, wherein the antiviral therapy comprises
nucleoside/nucleotide therapy or immunotherapy.
30. The method of claim 29, wherein the nucleoside/nucleotide therapy is selected from among lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy.
31. The use of claim 29, wherein the immunotherapy therapy comprises interferon-alpha (IFN-a) therapy.
32. The method of any one of claims 28 to 31, further comprising providing a recommendation to the subject to continue antiviral therapy if it is determined that HBV recrudescence is likely.
33. The method of any one of claims 28 to 31, further comprising providing a recommendation to the subject to terminate antiviral therapy if it is determined that HBV recrudescence is not likely.
34. Use of an antiviral therapy for treating HBV infection in a subject, wherein the subject is exposed to the antiviral therapy on the basis of a determination that HBV recrudescence is likely according to any one of claims 1 to 32.
35. The use of claim 34, wherein the antiviral therapy comprises nucleoside/nucleotide therapy or immunotherapy.
36. The use of claim 35, wherein the nucleoside/nucleotide therapy is selected from among lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine and tenofovir therapy.
37. The use of claim 35, wherein the immunotherapy comprises interferon-alpha (IFN-α) therapy.
38. A method for treating HBV infection in a subject, comprising :
performing the method of any one of claims 1 to 31 ; and
exposing the subject to an antiviral therapy if it is determined that HBV recrudescence is likely.
39. A method for treating HBV infection in a subject, comprising :
(a) sending a biological sample obtained from a subject to a laboratory to
(i) conduct the method of any one of claims 1 to 24 and 27 to 31; and
(ii) provide the results of the method, wherein the results comprise a determination of whether HBV recrudescence is likely or unlikely;
(b) receiving the results from step (a); and
(c) exposing the subject to an antiviral therapy if the results comprise a determination that HBV recrudescence is likely.
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