Understanding Early Serum Hepatitis D Virus and Hepatitis B Surface Antigen Kinetics During Pegylated Interferon-alpha Therapy Via Mathematical Modeling Jeremie Guedj,1,2,3 Yaron Rotman,4 Scott J. Cotler,5 Christopher Koh,4 Peter Schmid,6 Jeff Albrecht,6 Vanessa Haynes-Williams,4 T. Jake Liang,4 Jay H. Hoofnagle,4 Theo Heller,4 and Harel Dahari1,5 There is little information on the early kinetics of hepatitis delta virus (HDV) and hepatitis B surface antigen (HBsAg) during interferon-a therapy. Here a mathematical model was developed and fitted to frequent HDV and HBsAg kinetic data from 10 patients during the first 28 weeks of pegylated-interferon-a2a (peg-IFN) therapy. Three patients achieved a complete virological response (CVR), defined as undetectable HDV 6 months after treatment stopped with loss of HBsAg and anti-HBsAg seroconversion. After initiation of therapy, a median delay of 9 days (interquartile range [IQR]: 5-15) was observed with no significant changes in HDV level. Thereafter, HDV declined in a biphasic manner, where a rapid first phase lasting for 25 days (IQR: 23-58) was followed by a slower or plateau second phase. The model predicts that the main effect of peg-IFN is to reduce HDV production/release with a median effectiveness of 96% (IQR: 93-99.8). Median serum HDV half-life (t1/2) was estimated as 2.9 days (IQR: 1.5-5.3) corresponding to a pretreatment production and clearance of about 1010 (IQR: 109.7-1010.7) virions/day. None of the patients with flat second phase in HDV achieved CVR. HBsAg kinetics of decline paralleled the second phase of HDV decline consistent with HBsAg-productive-infected cells being the main source of production of HDV, with a median t1/2 of 135 days (IQR: 20-460). The interferon lambda-3 polymorphism (rs12979860) was not associated with kinetic parameters. Conclusion: Modeling results provide insights into HDV-host dynamics, the relationship between serum HBsAg levels and HBsAg-infected cells, IFN’s mode of action, and its effectiveness. The observation that a flat second phase in HDV and HBsAg kinetics was associated with failure to achieve CVR provides the basis to develop early stopping rules during peg-IFN treatment in HDV-infected patients. (HEPATOLOGY 2014;60:1902-1910)
H
epatitis D virus (HDV) is a single-stranded, circular RNA genome that was identified in 1980 as an infectious agent causing hepatitis in persons who are infected with hepatitis B virus (HBV).1 HDV infection is the most severe form of chronic viral hepatitis in humans,2-4 with an accelerated course of liver disease compared to chronic monoinfection with HBV.5 It is estimated that 15-20 million persons worldwide are chronically infected with HDV, i.e., about 5% of the
HBV-infected population.6 Hepatitis B surface antigen (HBsAg) encapsidates HDV RNA, forming the viral envelope.7 As such, HDV is a defective virus that depends on HBV envelope protein to enter hepatocytes and assemble new HDV particles. At least eight different HDV genotypes have been described,8 with genotype 1 being the most common in many areas of the world. Treatment of HDV infection has been notoriously difficult. Treatment with interferon (IFN)-based
Abbreviations: CVR, complete virological response; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDV, hepatitis delta virus; peg-IFN, pegylatedinterferon-a2a. From the 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM, USA; 2IAME, UMR 1137, INSERM, F-75018, Paris, e , F-75018, Paris, France; 4Liver Diseases Branch, NIDDK, NIH, Bethesda, MD, France; 3IAME, UMR 1137, Univ Paris 7, Diderot, Sorbonne Paris Cit 5 USA; Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Loyola University Medical Center, Maywood, IL, USA; 6National Genetics Institute, Los Angeles, CA, USA. Received February 7, 2014; accepted August 5, 2014. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27357/suppinfo. The clinical study was supported by the intramural research program of NIDDK. Portions of this work were performed under the auspices of the U.S. Department of Energy under contract DE-AC52-06NA25396 and supported by NIH grant P20-GM103452. 1902
HEPATOLOGY, Vol. 60, No. 6, 2014
therapies can achieve HDV RNA negativity in 25% of patients.9-12 However, the more definite and durable endpoint of HDV RNA negativity together with loss of HBsAg, equivalent to HDV eradication, is even more difficult to achieve. Monotherapy with nucleos/ tide analogs such as adefovir, lamivudine, or ribavirin are not effective in treating HDV and have not been shown to significantly improve complete virological response (CVR) rates when combined with pegylated (peg)-IFN compared to peg-IFN alone.10,12,13 Mathematical modeling of viral kinetics aims to understand and quantify the biological mechanisms that govern the changes in the viral load and related biomarkers that occur with antiviral therapy. Mathematical modeling provided estimates of key viral and host parameters in viral infections including HIV,14-16 HBV,17-19 and HCV20 as well as giving valuable insight into the modes of action of antiviral agents. For HCV, which like HDV is a single-stranded RNA virus that causes chronic infection and can be eradicated by treatment, viral kinetic parameters showed a high predictive ability for treatment outcome and can be used to individualize treatment duration.21-24 Here we analyzed data from a clinical trial in which patients with chronic HBV and HDV were treated for up to 260 weeks with peg-IFN. Frequent blood samples were obtained in the first 28 weeks following initiation of peg-IFN, allowing for precise characterization of the early dynamics of serum HDV RNA and HBsAg in treated patients. A dual model for HDV and HBsAg was developed to estimate key parameters of HDV and HBsAg dynamics, including peg-IFN effectiveness in blocking HDV production and/or release from infected cells, serum HDV half-life, and HBsAg-infected cell half-life.
Patients and Methods Below is a brief description of patients and methods. More details on patients, study design, virological, and genetic assays can be found in Heller et al.25 Patients. Data were obtained from 12 patients (out of 13), with confirmed HDV genotype 1, who participated in a clinical trial (#NCT00023322) of long-term
GUEDJ ET AL.
1903
treatment (up to 260 weeks) of HDV with pegylated IFNa-2a (peg-IFN). Baseline characteristics of the study population are provided in Table 1. Patients were treated with a fixed dose of 180 lg of peg-IFN monotherapy weekly during the first 28 weeks of therapy, following which dose increases or reductions (for efficacy or safety, respectively) were allowed.25 Two patients had a treatment interruption, between week 4 and 10 (Patient 1) and between week 19 and 21 (Patient 8). Virological and Genetic Assays. HDV-RNA levels were measured by a real-time polymerase chain reaction (PCR) assay (National Genetics Institute, Los Angeles, CA) with a lower limit of quantification (LOQ) of 100 genome equivalents per mL (GE/mL). HBsAg levels were measured with the Roche Cobas HBsAg quantitative assay, with a lower limit of detection of 0.055 IU/ mL. The interferon lambda 3, IFNL3 (formerly called IL28B) associated single-nucleotide polymorphism (SNP) rs12979860 was determined in 10 patients using TaqMan genotyping assay (Table 1). Blood Samples. Frequent HDV-RNA, HBVDNA, alanine aminotransferase (ALT), and HBsAg levels were measured at times 0, 6, 12, and 18 hours during the first day of peg-IFN treatment, at days 2, 3 and 7, and at weeks 2, 3, 4, 8, 12, 16, 20, 24, and 28. The viral kinetic analysis was limited to the first 28 weeks of treatment. Mathematical Modeling of HDV and HBsAg Kinetics. Serum HDV RNA levels declined during the first 28 weeks of peg-IFN therapy in a biphasic manner similar to what has been observed during antiviral therapy in some HBV-monoinfected patients19,26 and HCV-infected patients22,27 treated with IFN-based therapy. In addition, serum HBsAg kinetics were associated with the second phase kinetics of serum HDV RNA (see Supporting Material and Results). Based on these observations we developed a dual model that coupled HDV RNA and HBsAg kinetics: 8 dI =dt5bð12hÞVTo 2dI > > < dV =dt5pðI 2eÞI 2cV (1) > > : dH =dt5pH I 2cH H
Address reprint requests to: Harel Dahari, Ph.D., Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Loyola University Medical Center, Maywood, IL, USA. E-mail: [email protected] or Theo Heller, M.D., Liver Diseases Branch, NIDDK, NIH, Bethesda, MD. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is Copyright V in the public domain in the USA. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27357 Potential conflict of interest: Nothing to report.
1904
GUEDJ ET AL.
HEPATOLOGY, December 2014
Table 1. Baseline Patient Characteristics
1* 2* 3 4 5* 6† 8† 9 10 11 12 13 Mean 6 SD
Gender
Race
rs12979860 Genotype
F M F M M M M M M M M M
C C C C AA C C C C C C C
CC — — CT CT CT CC CT CC CC CT CC
Liver Stage
Age (Years)
Time Infected (Years)
Baseline HDV RNA (logGE/mL)
3 3 3 6 3 4 3 4 3 3 4 6
53 45 32 44 48 58 20 47 46 44 18 49 42 6 12
27 28 26 35 UN 34 UN UN 25 7 UN 14 24 6 9
4.8 6.9 6.8 6.2 6.8 4.5 7.3 6.5 6.9 8.0 7.6 8.5 6.7 6 1.1
HBeAg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos Pos
Baseline ALT (Fold UNL)
Baseline HBsAg (LogIU/mL)
Baseline HBV DNA (Log IU/mL)
Treatment Duration (Months)
41(2.2) 48 (1.6) 31 (1.6) 296 (9.9) 206 (6.9) 42 (1.4) 84 (2.8) 506 (16.9) 107 (3.6) 59 (2.0) 185 (6.2) 172 (5.7) 148 (5.1) 6 134 (4.4)
2.9 3.2 4.2 4.2 4.0 2.6 4.3 3.8 4.3 4.0 4.3 3.5 3.8 6 0.6
3.5 UD UD 0.1 log/mL/week, two achieved CVR after less than a year of treatment (Patients 1 and 2). This rapid second phase of viral decline was mostly due to a high loss rate of infected cells rather than to a high antiviral effectiveness (Table 2). Interestingly, three patients (Patients 8, 9, and 12) had greater than 99% effectiveness in blocking viral production (see parameter e in Table 2) but yet did not achieve a rapid second phase of viral decline or CVR, which reflects the lack of association between e and k2 (or d; not shown). None of the five patients with extremely slow (or flat) second phase slope of HDV, i.e., k2
H
epatitis D virus (HDV) is a single-stranded, circular RNA genome that was identified in 1980 as an infectious agent causing hepatitis in persons who are infected with hepatitis B virus (HBV).1 HDV infection is the most severe form of chronic viral hepatitis in humans,2-4 with an accelerated course of liver disease compared to chronic monoinfection with HBV.5 It is estimated that 15-20 million persons worldwide are chronically infected with HDV, i.e., about 5% of the
HBV-infected population.6 Hepatitis B surface antigen (HBsAg) encapsidates HDV RNA, forming the viral envelope.7 As such, HDV is a defective virus that depends on HBV envelope protein to enter hepatocytes and assemble new HDV particles. At least eight different HDV genotypes have been described,8 with genotype 1 being the most common in many areas of the world. Treatment of HDV infection has been notoriously difficult. Treatment with interferon (IFN)-based
Abbreviations: CVR, complete virological response; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDV, hepatitis delta virus; peg-IFN, pegylatedinterferon-a2a. From the 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM, USA; 2IAME, UMR 1137, INSERM, F-75018, Paris, e , F-75018, Paris, France; 4Liver Diseases Branch, NIDDK, NIH, Bethesda, MD, France; 3IAME, UMR 1137, Univ Paris 7, Diderot, Sorbonne Paris Cit 5 USA; Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Loyola University Medical Center, Maywood, IL, USA; 6National Genetics Institute, Los Angeles, CA, USA. Received February 7, 2014; accepted August 5, 2014. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27357/suppinfo. The clinical study was supported by the intramural research program of NIDDK. Portions of this work were performed under the auspices of the U.S. Department of Energy under contract DE-AC52-06NA25396 and supported by NIH grant P20-GM103452. 1902
HEPATOLOGY, Vol. 60, No. 6, 2014
therapies can achieve HDV RNA negativity in 25% of patients.9-12 However, the more definite and durable endpoint of HDV RNA negativity together with loss of HBsAg, equivalent to HDV eradication, is even more difficult to achieve. Monotherapy with nucleos/ tide analogs such as adefovir, lamivudine, or ribavirin are not effective in treating HDV and have not been shown to significantly improve complete virological response (CVR) rates when combined with pegylated (peg)-IFN compared to peg-IFN alone.10,12,13 Mathematical modeling of viral kinetics aims to understand and quantify the biological mechanisms that govern the changes in the viral load and related biomarkers that occur with antiviral therapy. Mathematical modeling provided estimates of key viral and host parameters in viral infections including HIV,14-16 HBV,17-19 and HCV20 as well as giving valuable insight into the modes of action of antiviral agents. For HCV, which like HDV is a single-stranded RNA virus that causes chronic infection and can be eradicated by treatment, viral kinetic parameters showed a high predictive ability for treatment outcome and can be used to individualize treatment duration.21-24 Here we analyzed data from a clinical trial in which patients with chronic HBV and HDV were treated for up to 260 weeks with peg-IFN. Frequent blood samples were obtained in the first 28 weeks following initiation of peg-IFN, allowing for precise characterization of the early dynamics of serum HDV RNA and HBsAg in treated patients. A dual model for HDV and HBsAg was developed to estimate key parameters of HDV and HBsAg dynamics, including peg-IFN effectiveness in blocking HDV production and/or release from infected cells, serum HDV half-life, and HBsAg-infected cell half-life.
Patients and Methods Below is a brief description of patients and methods. More details on patients, study design, virological, and genetic assays can be found in Heller et al.25 Patients. Data were obtained from 12 patients (out of 13), with confirmed HDV genotype 1, who participated in a clinical trial (#NCT00023322) of long-term
GUEDJ ET AL.
1903
treatment (up to 260 weeks) of HDV with pegylated IFNa-2a (peg-IFN). Baseline characteristics of the study population are provided in Table 1. Patients were treated with a fixed dose of 180 lg of peg-IFN monotherapy weekly during the first 28 weeks of therapy, following which dose increases or reductions (for efficacy or safety, respectively) were allowed.25 Two patients had a treatment interruption, between week 4 and 10 (Patient 1) and between week 19 and 21 (Patient 8). Virological and Genetic Assays. HDV-RNA levels were measured by a real-time polymerase chain reaction (PCR) assay (National Genetics Institute, Los Angeles, CA) with a lower limit of quantification (LOQ) of 100 genome equivalents per mL (GE/mL). HBsAg levels were measured with the Roche Cobas HBsAg quantitative assay, with a lower limit of detection of 0.055 IU/ mL. The interferon lambda 3, IFNL3 (formerly called IL28B) associated single-nucleotide polymorphism (SNP) rs12979860 was determined in 10 patients using TaqMan genotyping assay (Table 1). Blood Samples. Frequent HDV-RNA, HBVDNA, alanine aminotransferase (ALT), and HBsAg levels were measured at times 0, 6, 12, and 18 hours during the first day of peg-IFN treatment, at days 2, 3 and 7, and at weeks 2, 3, 4, 8, 12, 16, 20, 24, and 28. The viral kinetic analysis was limited to the first 28 weeks of treatment. Mathematical Modeling of HDV and HBsAg Kinetics. Serum HDV RNA levels declined during the first 28 weeks of peg-IFN therapy in a biphasic manner similar to what has been observed during antiviral therapy in some HBV-monoinfected patients19,26 and HCV-infected patients22,27 treated with IFN-based therapy. In addition, serum HBsAg kinetics were associated with the second phase kinetics of serum HDV RNA (see Supporting Material and Results). Based on these observations we developed a dual model that coupled HDV RNA and HBsAg kinetics: 8 dI =dt5bð12hÞVTo 2dI > > < dV =dt5pðI 2eÞI 2cV (1) > > : dH =dt5pH I 2cH H
Address reprint requests to: Harel Dahari, Ph.D., Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Loyola University Medical Center, Maywood, IL, USA. E-mail: [email protected] or Theo Heller, M.D., Liver Diseases Branch, NIDDK, NIH, Bethesda, MD. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is Copyright V in the public domain in the USA. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27357 Potential conflict of interest: Nothing to report.
1904
GUEDJ ET AL.
HEPATOLOGY, December 2014
Table 1. Baseline Patient Characteristics
1* 2* 3 4 5* 6† 8† 9 10 11 12 13 Mean 6 SD
Gender
Race
rs12979860 Genotype
F M F M M M M M M M M M
C C C C AA C C C C C C C
CC — — CT CT CT CC CT CC CC CT CC
Liver Stage
Age (Years)
Time Infected (Years)
Baseline HDV RNA (logGE/mL)
3 3 3 6 3 4 3 4 3 3 4 6
53 45 32 44 48 58 20 47 46 44 18 49 42 6 12
27 28 26 35 UN 34 UN UN 25 7 UN 14 24 6 9
4.8 6.9 6.8 6.2 6.8 4.5 7.3 6.5 6.9 8.0 7.6 8.5 6.7 6 1.1
HBeAg
Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos Pos
Baseline ALT (Fold UNL)
Baseline HBsAg (LogIU/mL)
Baseline HBV DNA (Log IU/mL)
Treatment Duration (Months)
41(2.2) 48 (1.6) 31 (1.6) 296 (9.9) 206 (6.9) 42 (1.4) 84 (2.8) 506 (16.9) 107 (3.6) 59 (2.0) 185 (6.2) 172 (5.7) 148 (5.1) 6 134 (4.4)
2.9 3.2 4.2 4.2 4.0 2.6 4.3 3.8 4.3 4.0 4.3 3.5 3.8 6 0.6
3.5 UD UD 0.1 log/mL/week, two achieved CVR after less than a year of treatment (Patients 1 and 2). This rapid second phase of viral decline was mostly due to a high loss rate of infected cells rather than to a high antiviral effectiveness (Table 2). Interestingly, three patients (Patients 8, 9, and 12) had greater than 99% effectiveness in blocking viral production (see parameter e in Table 2) but yet did not achieve a rapid second phase of viral decline or CVR, which reflects the lack of association between e and k2 (or d; not shown). None of the five patients with extremely slow (or flat) second phase slope of HDV, i.e., k2
