Therapeutic strategies for recent virus in the era of direct-acting antiviral therapy

Marianne Martinello

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

The Kirby Institute Faculty of Medicine

May 2017

Page 1 of 309 Thesis/Dissertation Sheet

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Martinello First name: Marianne Abbreviation for degree as given in the University calendar: PhD School: Kirby Institute Faculty: Medicine Title: Therapeutic strategies for recent hepatitis C virus infection in the era of direct-acting antiviral therapy

Abstract: Background: The management of recent hepatitis C virus (HCV) infection is uncertain, as the therapeutic landscape evolves with the advent of direct-acting antiviral (DAA) therapy.

Aims: The broad aim of this research was to examine novel therapeutic strategies in recent HCV following the availability of DAA therapy. Specific aims included assessing the feasibility, efficacy and safety of response-guided (IFN) -containing and ultra-short IFN-free therapy; calculating reinfection incidence following treatment; and determining the clinical significance of drug-drug interactions in HIV/HCV co-infection.

Methods: In Chapters Two and Three, the efficacy and safety of short and/or response-guided treatment among individuals with recent HCV was examined in three open-label trials: ATAHC II (n=52; pegylated-IFN +/- ), DARE-C I (n=14; pegylated-IFN, ribavirin + ) and DARE-C II (n=19; + ribavirin). In Chapter Four, reinfection incidence following treatment for recent HCV was calculated among those who achieved an end-of-treatment response in ATAHC I and II, DARE-C I and II (n=120). In Chapter Five, a simulation of potential drug-drug interactions between combination antiretroviral therapy and DAAs was performed in those with HIV/HCV enrolled in CEASE-D (n=257).

Page 2 of 309 Key Findings: A total of 85 individuals (73% HIV) with recent HCV commenced short and/or response-guided treatment between 2010 and 2015; none of the strategies trialled was optimal. Given toxicity, the applicability of an IFN-containing strategy is limited. Conversely, six weeks of sofosbuvir and ribavirin was safe and well tolerated, but efficacy was poor. High reinfection incidence, particularly in individuals reporting injection drug use following treatment, was seen. These trials confirmed the feasibility of short duration therapy in recent HCV and informed contemporary trial design. Optimal DAA regimen choice for future research will be crucial; the combination of a nucleotide analogue and an NS5A inhibitor appears appropriate given potency and little potential for drug-drug interactions in HIV/HCV co-infection.

Conclusion: The role and efficacy of ultra-short duration therapy in recent HCV requires further evaluation with potent DAA regimens. The significant risk for HCV reinfection following treatment in individuals with ongoing behaviour facilitating transmission emphasises the need for post-treatment surveillance, harm reduction strategies and education.

Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

14 Dec 2016

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The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

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Page 3 of 309 Contents Thesis/Dissertation Sheet ...... 2 Supervisor Statement ...... 8 Originality Statement ...... 9 Copyright Statement ...... 10 Authenticity Statement ...... 11 Acknowledgements ...... 12 List of publications and presentations ...... 14 Publications ...... 14 Conference presentations ...... 15 Abbreviations ...... 17 Chapter 1 Introduction and Literature Review...... 19 Epidemiology of recent HCV infection ...... 19 HCV incidence and risk factors for acquisition ...... 20 Detection of acute HCV infection ...... 27 Management of acute and recent HCV infection ...... 29 Natural history and spontaneous clearance ...... 29 Timing of HCV treatment initiation ...... 30 Interferon-based treatment of acute HCV mono-infection ...... 30 Interferon-based treatment of recent HCV infection in PWID ...... 32 Interferon-based treatment of recent HCV infection in HIV co-infection ...... 32 Duration of interferon-based treatment in recent HCV infection ...... 33 Evidence gaps related to the treatment of acute and recent HCV infection ...... 33 Overview of Protease, Polymerase and Assembly Inhibitors for the Treatment of HCV...... 35 Mechanism of drug action...... 38 Mode of drug administration and dosage ...... 42 Adverse reactions and toxicity ...... 46 Direct-acting antiviral efficacy in chronic HCV infection among PWID and people with HIV/HCV coinfection ...... 52 Evidence from clinical trials ...... 52 Evidence from “real world” cohorts ...... 57 Reinfection following HCV treatment ...... 58 Who is at “high-risk” of reinfection? Defining the population of interest ...... 58 HCV reinfection among people who inject drugs ...... 61 HCV reinfection among HIV-positive MSM ...... 62 Mathematical modelling: Treatment-as-Prevention and the impact of reinfection ...... 63 Thesis rationale and objectives ...... 65 Chapter 2 The efficacy, safety and feasibility of response-guided interferon-based therapy in recent HCV infection ...... 67 Chapter Introduction and Objectives ...... 67

Page 4 of 309 Declaration ...... 69 Co-authorship Acknowledgement...... 70 Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II and DARE-C I studies ...... 71 Abstract ...... 72 Introduction ...... 73 Methods ...... 74 Results ...... 78 Discussion ...... 89 Supplementary Material ...... 91 Methods...... 91 Rationale for response-guided interferon-based therapy allocation ...... 95 Tables ...... 96 Figures...... 101 Chapter 3 The efficacy, safety and feasibility of ultra-short duration interferon-free therapy in recent HCV infection ...... 103 Chapter Introduction and Objectives ...... 103 Declaration ...... 104 Co-authorship Acknowledgement...... 105 Sofosbuvir and ribavirin for six weeks is not effective among people with recent HCV infection: The DARE-C II study...... 106 Abstract ...... 107 Introduction ...... 108 Methods ...... 109 Results ...... 113 Discussion ...... 122 Supplementary Material ...... 125 Methods...... 125 Tables ...... 127 Figures...... 130 Chapter 4 HCV reinfection incidence among individuals treated for recent infection ...... 136 Chapter Introduction and Objectives ...... 136 Declaration ...... 137 Co-authorship Acknowledgement...... 138 HCV reinfection incidence among individuals treated for recent infection...... 139 Abstract ...... 140 Introduction ...... 141 Methods ...... 142 Results ...... 145 Discussion ...... 154

Page 5 of 309 Supplementary Material ...... 158 Methods...... 158 Tables ...... 160 Figures...... 167 Chapter 5 Optimising direct-acting antiviral regimen choice in HIV/HCV co-infection – informing future trial design ...... 168 Chapter Introduction and Objectives ...... 168 Declaration ...... 169 Co-authorship Acknowledgement...... 170 Antiretroviral use in the CEASE cohort study and implications for DAA therapy in HIV/HCV co-infection...... 171 Abstract ...... 172 Introduction ...... 173 Methods ...... 174 Results ...... 177 Discussion ...... 184 Supplementary Material ...... 187 Tables ...... 187 Figures...... 190 Chapter 6 Integrated Discussion ...... 192 Key Findings ...... 192 Aim 1: To evaluate the efficacy and safety of response-guided interferon-containing therapy in individuals with recent HCV infection ...... 192 Aim 2: To evaluate the feasibility, efficacy and safety of ultra-short duration interferon- free therapy in individuals with recent HCV infection ...... 194 Aim 3: To evaluate the efficacy of response-guided interferon-containing and short duration interferon-free therapy in HCV mono-infection as compared with HIV/HCV co- infection ...... 195 Aim 4: To evaluate treatment adherence in individuals receiving response-guided interferon-containing and short duration interferon-free therapy with recent HCV infection ...... 195 Aim 5: To calculate the incidence of HCV reinfection among individuals treated for recent HCV infection...... 196 Aim 6: To assess the clinical significance of drug-drug interactions between HCV DAAs and HIV antiretroviral therapy...... 197 Implications for the findings ...... 199 Directions for future research ...... 202 Thesis strengths and limitations ...... 208 Conclusion ...... 210 References ...... 211 Supplementary Appendix ...... 239 Investigational direct-acting antivirals for the treatment of HCV infection...... 240

Page 6 of 309 Chemical structure of individual approved HCV direct-acting antivirals...... 247 Antiviral activity of approved HCV direct-acting antivirals ...... 254 Pharmacokinetics of approved HCV direct-acting antivirals ...... 270 High adherence to short duration response-guided treatment among people with recent HCV infection: The ATAHC II and DARE-C I studies ...... 296 List of investigators ...... 308

Page 7 of 309 Supervisor Statement

Statement by Gail Matthews

I hereby certify that all co-authors of the published or submitted papers agree to Marianne Martinello submitting those papers as part of her Doctoral Thesis.

Signed

Date 14 Dec 2016

Statement by Gregory Dore

I hereby certify that all co-authors of the published or submitted papers agree to Marianne Martinello submitting those papers as part of her Doctoral Thesis.

Signed

Date 14 Dec 2016

Page 8 of 309 Originality Statement

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………...... Date 14 Dec 2016

Page 9 of 309 Copyright Statement

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

Signed ……………………………………………...... Date 14 Dec 2016

Page 10 of 309 Authenticity Statement

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

Signed ……………………………………………...... Date 14 Dec 2016

Page 11 of 309 Acknowledgements

The contributions and support of many people have made this thesis possible.

I am indebted to my supervisors, Associate Professor Gail Matthews and Professor Gregory Dore, for their advice, support and mentorship. They have provided with numerous opportunities in my research and clinical career for which I am very grateful.

I would like to thank everyone who has contributed to the ATAHC I, ATAHC II, DARE-C I and DARE-C II studies, including all protocol steering committee members, staff at co- ordinating centres, site investigators, site co-ordinators and data managers.

In regard to ATAHC II and DARE-C I, I would specifically like to acknowledge the following people for their contributions: Gail Matthews, Gregory Dore, Margaret Hellard, David Shaw, Kathy Petoumenos, Tanya Applegate, Jason Grebely, David Iser, Andrew Lloyd, Alex Thompson, Joe Sasadeusz and Paul Haber for the initial study concept and design; Gail Matthews, Gregory Dore, Margaret Hellard, David Shaw, David Iser and Joe Sasadeusz for data acquisition; Gail Matthews and Gregory Dore for their assistance with data analysis and interpretation; Kathy Petoumenos for assistance with statistical analysis; Barbara Yeung and Laurence Maire for study management, administrative and technical support; and all of those listed for their critical review of the final manuscript.

In regard to DARE-C II, I would specifically like to acknowledge the following people for their contributions: Gail Matthews, Gregory Dore, Edward Gane, Margaret Hellard, David Shaw, Kathy Petoumenos, Tanya Applegate, Jason Grebely, Pip Marks and Joe Sasadeusz for the initial study concept and design; Gail Matthews, Gregory Dore, Edward Gane, Margaret Hellard, David Shaw and Joe Sasadeusz for data acquisition; Kathy Petoumenos for assistance with statistical analysis; Gail Matthews, Gregory Dore and Jason Grebely for their assistance with data analysis and interpretation; Barbara Yeung and Laurence Maire for study management, administrative and technical support; and all of those listed for their critical review of the final manuscript.

Additionally, I would like to acknowledge the contributions of Francois Lamoury and Danica Martinez, Kirby Institute, UNSW, Sydney for their assistance with RNA extraction, sequencing and analysis and Mohammed Eslam, Westmead Hospital, Sydney for his assistance with interferon-lambda single nucleotide polymorphism analysis. I would like to thank Ana

Page 12 of 309 Schteinman and Richard Day, Department of Pharmacology, St Vincent’s Hospital, Sydney, for performing therapeutic drug monitoring and quantifying ribavirin plasma concentrations. I would like to acknowledge Mahshid Tamaddoni, Kirby Institute, UNSW, Sydney, for her assistance with data management. I would also like to acknowledge the assistance of the following site study coordinators for their long-standing and continuing commitment to clinical research: Angelle Lockie, Victoria Oliver and Amy Cole, Auckland City Hospital, New Zealand; Sally von Bibra, The Alfred Hospital, VIC; Catherine Ferguson, Royal Adelaide Hospital, SA; Joanne Patterson and Kim Crook, Royal Melbourne Hospital, VIC; Rebecca Hickey, St Vincent’s Hospital, NSW.

I would like to thank everyone who has contributed and continues to contribute to the CEASE project, including all protocol steering committee members, staff at coordinating centres, site investigators, site co-ordinators and data managers. Specifically, I would like to acknowledge the following people for their contribution to the included manuscript: Gail Matthews and Gregory Dore for the initial study concept and design; Gail Matthews, Gregory Dore, Rohan Bopage, Robert Finlayson and Mark Bloch for data acquisition; Gail Matthews and Gregory Dore for their assistance in data analysis and interpretation; Jasmine Skurowski for study management, administrative and technical support; and all of those listed for their critical review of the final manuscript. Additionally, I would like to acknowledge the contributions of Arlen Wilcox, Kirby Institute, UNSW, Sydney, for his assistance with study co-ordination and Ecaterina Filep, Kirby Institute, UNSW, Sydney, for her assistance with data management.

Most importantly, I would like to thank all of the study participants and their families, without whom, this research would not have been possible.

Finally, I would like to thank my family and closest friends for their continual support, care and unwavering optimism.

Page 13 of 309 List of publications and presentations

Publications A list of peer-reviewed publications (i.e., accepted or published) related to the work contained within this thesis is presented below: • Martinello M, Matthews GV. Enhancing the detection and management of acute hepatitis C virus infection. Int J Drug Policy. 2015 Oct; 26(10):899-910. • Martinello M, Schteinman A, Alavi M, Williams K, Dore GJ, Day R, Matthews GV. The impact of ribavirin plasma concentration on the efficacy of the interferon-sparing regimen, sofosbuvir and ribavirin. Antivir Ther. 2016; 21(2):127-32. • Martinello M, Hellard M, Shaw D, Petoumenos K, Applegate T, Grebely J, Yeung B, Maire L, Iser D, Lloyd A, Thompson A, Sasadeusz J, Haber P, Dore GJ, Matthews GV. Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II and DARE-C I studies. Antivir Ther. 2016;21(5):425-34. doi: 10.3851/IMP3035. • Martinello M, Dore GJ. Editorial Commentary: Interferon-free Hepatitis C Treatment Efficacy From Clinical Trials Will Translate to "Real World" Outcomes. Clin Infect Dis. 2016 Apr 1;62(7):927-8. • Martinello M, Amin J, Matthews GV, Dore GJ. Prevalence and Disease Burden of HCV Coinfection in HIV Cohorts in the Asia Pacific Region: A Systematic Review and Meta-Analysis. AIDS Rev. 2016;18(2):68-80. • Martinello M, Dore GJ, Skurowski J, Bopage RI, Finlayson R, Baker D, Bloch M, Matthews GV. Antiretroviral use in the CEASE cohort study and implications for DAA therapy in HIV/HCV co-infection. Open Forum Infectious Diseases. 2016 May 18;3(2): ofw105. doi: 10.1093/ofid/ofw105. • Martinello M, Gane E, Hellard M, Sasadeusz J, Shaw D, Petoumenos K, Applegate T, Grebely J, Maire L, Marks P, Dore GJ, Matthews GV. Sofosbuvir and ribavirin for six weeks is not effective among people with recent HCV infection: The DARE-C II study. Hepatology. 2016; 64(6):1911-1921. • Martinello M, Hajarizadeh B, Grebely J, Dore GJ, Matthews GV. HCV cure and reinfection among people with HIV/HCV co-infection and people who inject drugs. Curr HIV/AIDS Rep. 2017;14:110-121. • Martinello M, Grebely J, Petoumenos K, Gane E, Hellard M, Shaw D, Sasadeusz J, Applegate T, Dore GJ, Matthews GV. HCV reinfection incidence among individuals treated for recent infection. J Viral Hepatitis. 2017;24:359-370.

Page 14 of 309 Conference presentations A list of conference presentations (i.e., accepted or published) related to the work contained within this thesis is presented below: • Martinello M, Hellard M, Shaw D, Petoumenos K, Applegate T, Grebely J, Yeung B, Maire L, Iser D, Lloyd A, Thompson A, Sasadeusz J, Haber P, Dore GJ, Matthews GV. Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II study [#957]. Poster session presented at: American Association for the Study of Liver Disease; 2014 November 7 - 11; Boston, MA, USA. “Presidential Poster of Distinction”. • Martinello M, Schteinman A, Alavi M, Williams K, Dore GJ, Day R, Matthews GV. The impact of ribavirin plasma concentration on the efficacy of the interferon-sparing regimen, sofosbuvir and ribavirin [#997]. Poster session presented at: American Association for the Study of Liver Disease; 2014 November 7 - 11; Boston, MA, USA. • Martinello M, Grebely J, Shaw D, Hellard M, Petoumenos K, Applegate T, Yeung B, Maire L, Iser D, Lloyd A, Sasadeusz J, Dore GJ, Matthews GV. High adherence to short duration response-guided treatment among people with recent HCV infection: The ATAHC II and DARE-C I studies [#112]. Poster session presented at: 4th International Symposium on Hepatitis Care in Substance Users; 2015 October 7-9; Sydney, NSW, Australia. • Martinello M, Gane E, Hellard M, Sasadeusz J, Shaw D, Petoumenos K, Applegate T, Grebely J, Maire L, Marks P, Cooper D, Dore GJ, Matthews GV. Sofosbuvir and ribavirin for six weeks is not effective among people with acute and recently acquired HCV infection: The DARE-C II study [#1083]. Poster session presented at: American Association for the Study of Liver Disease; 2015 November 13 - 17; San Francisco, CA, USA. • Martinello M, Dore GJ, Skurowski J, Bopage RI, Finlayson R, Baker D, Bloch M, Matthews GV. Antiretroviral use in the CEASE cohort study and implications for DAA therapy in HIV/HCV co-infection [#451]. Poster session presented at: Conference on Retroviruses and Opportunistic ; 2016 February 22 – 25; Boston, MA, USA. • Martinello M, Dore GJ, Skurowski J, Amin J, Bopage RI, Finlayson R, Baker D, Bloch M, Matthews GV. Sexual behaviour is associated with recently acquired HCV in HIV/HCV co-infected MSM [#545]. Poster session presented at: Conference on Retroviruses and Opportunistic Infections; 2016 February 22 – 25; Boston, MA, USA.

Page 15 of 309 • Martinello M, Petoumenos K, Grebely J, Gane E, Hellard M, Shaw D, Sasadeusz J, Applegate T, Yeung B, Maire L, Dore GJ, Matthews GV. Incidence of HCV reinfection among treated individuals with recently acquired infection [#FRI-184]. Poster session presented at: The International Liver Congress - EASL; 2016 April 13- 17; Barcelona, Spain.

Page 16 of 309 Abbreviations (in alphabetical order)

ALT alanine aminotransferase AST aspartate aminotransferase ATAHC II Australian Trial in Acute Hepatitis C II BLoD below the limit of detection cART combination antiretroviral therapy CEASE Control and Elimination within Australia of Hepatitis C from people living with HIV CI confidence interval CYP cytochrome P450 DAA direct-acting antiviral DARE-C I Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C I DARE-C II Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C II DDI drug-drug interactions EC effective concentration ETR end-of-treatment response FC fold-change FDA Food and Drug Administration HCV hepatitis C virus HIV human immunodeficiency virus HPLC High-Performance Liquid Chromatography ICH-GCP International Conference on Harmonization Good Clinical Practice IDU injection drug use IFN interferon IFNL3/4 interferon-λ 3/4 IQR interquartile range ITT intention-to-treat LLoD lower limit of detection LLoQ lower limit of quantitation LTFU lost to follow up MSM men-who-have-sex-with-men NS non-structural NSP needle and syringe program OST opioid substitution therapy

Page 17 of 309 PEG-IFN pegylated interferon alfa-2a P-gp P-glycoprotein PrEP pre-exposure prophylaxis PWID people who inject drugs PWUD people who use drugs py person years RBV ribavirin RdRp RNA-dependent RNA polymerase RVR rapid virological response SAE serious adverse event SD standard deviation SNP single nucleotide polymorphism SVR sustained virological response TasP Treatment-as-Prevention TDF tenofovir disoproxil fumarate TDnq target detected, not quantifiable TND target not detected ULN upper limit of normal

Page 18 of 309 Chapter 1 Introduction and Literature Review

This chapter provides a background and rationale for the research presented in this thesis. It begins with an overview of the epidemiology and natural history of recent (acute and early chronic) hepatitis C virus (HCV) infection, focussing on populations at high risk of HCV transmission and acquisition, including people who inject drugs (PWID) and HIV-positive men- who-have-sex-with-men (MSM), followed by a summary of interferon-based management strategies for recent HCV infection. An overview of the clinical pharmacology and safety of HCV direct-acting antivirals (DAA) is included, preferentially discussing agents approved for use in chronic HCV infection, followed by a summary of DAA efficacy in chronic HCV infection among PWID and HIV-positive MSM. HCV reinfection following treatment-induced viral clearance comprises the fifth area of discussion. The chapter concludes with an overview of the thesis, including the thesis rationale, aims and key hypotheses.

Epidemiology of recent HCV infection Globally, an estimated 80 million people are living with chronic HCV infection, with 3-4 million HCV infections occurring annually (1, 2). Key at-risk populations for HCV acquisition include PWID and HIV-infected MSM (3, 4). Despite harm reduction strategies, such as needle and syringe programs (NSP) and opioid substitution therapy (OST), HCV transmission remains high among PWID (5, 6). The majority of new (60%) and existing (80%) infections in developed countries occur in this population with HCV antibody prevalence estimated at 67% (60-80%) (7, 8). Acquisition risk is greatest amongst younger injectors in the first years of unsafe injection practices (9, 10). HCV epidemics have also emerged among HIV-infected MSM, following broad uptake of HIV combination antiretroviral therapy (cART) (11, 12), although the burden of disease remains significantly lower than among PWID (13). Unsafe medical procedures continue to account for a large proportion of new HCV infections in low and middle income countries (13, 14).

Acute HCV infection refers to the 6 month period following infection acquisition, though definitions vary (15) and the distinction between acute and early chronic infection is somewhat arbitrary. Spontaneous clearance occurs in approximately 25% of those infected (16, 17), generally within six months, but can occur up to two years, following infection. The majority

Page 19 of 309 will develop chronic HCV infection and are at risk for cirrhosis (7-18% at 20 years) and hepatocellular carcinoma (1-3%) (18-21).

HCV incidence and risk factors for acquisition Detection of acute HCV infection has been hampered by its asymptomatic or non-specific presentation, lack of specific diagnostic tests and the inherent difficulties in identifying and following individuals at highest risk of transmitting and acquiring HCV. As such, epidemiological data on the incidence of acute HCV infection is somewhat limited.

Initial models of HCV incidence in the United States (US), based on the third National Health and Nutrition Examination Survey (NHANES III) (1988-1994), demonstrated considerable variation with a period of low incidence (0 - 44 per 100,000) before 1965, a transition period between 1965-1980 and a period of high incidence in the 1980s (100 - 200 per 100,000) (22). Surveillance data from the US Centres for Disease Control and Prevention (CDC) reported a decline in acute symptomatic HCV infection from 7.4 per 100,000 between 1982 – 1989 to 0.7 per 100,000 between 1994 - 2006, with the reduction in incidence related to blood donor screening and safer injecting practices in the era of HIV/AIDS, although PWID still accounted for 47% of these cases (23). Incidence fell further between 2006 and 2010 (2008 – 2010: 0.3 per 100,000), although subsequently rose to 0.6 per 100,000 in 2012 (24, 25). However, US surveillance relies upon passive reporting by health care providers and laboratories to state and local health departments (with the exception of 6 US jurisdictions funded for enhanced surveillance between 2006 – 2011) (25) thus leading to a marked underestimate of the true incidence. Mathematical modelling suggests that the incidence of new HCV infections is in the region of 5 - 10 times greater than reported. Recent estimates following enhanced surveillance suggest that 17,100 - 21,870 new infections occur annually in the US (approximately 12.3 infections per reported case) (25, 26).

In Europe, HCV incidence was estimated at 6.2 per 100,000 people in 2005 (27), though significant regional variation existed with many countries in Eastern Europe documenting a recent increase in injecting drug use with an associated rise in HCV incidence and prevalence (28).

In Australia, approximately 500 newly acquired cases are notified each year through national surveillance systems (29), with 90% of these cases occurring as a result of unsafe injecting practices (29, 30). Mathematical models estimate that new HCV infections peaked at 14,000 cases per annum in 1999 (31) before falling to 5,400 - 8,790 cases in 2013 (29, 32). The decline in incidence was largely attributed to a reduction in injection drug use.

Page 20 of 309 Although overall incidence is falling in many countries, high incidence persists in specific populations, including young adult (19.9 - 41.8 per 100 person years [py]) and incarcerated (5.5 - 31.6 per 100 py) PWID (Table 1-1, Table 1-2). In a recent meta-analysis, HCV incidence among prisoners with a history of injection drug use was 16.4 per 100 py (95% CI: 0.8, 32.1), considerably higher than that in the general incarcerated population (1.4 per 100 py; 95% CI: 0.1, 2.7) (33). Young adult PWID are at greatest risk of HCV acquisition, with recent onset injection drug use (<1 year) associated with a particularly high incidence (9). In 2 community cohorts of PWID in Sydney, Australia, HCV incidence in those aged 20 - 29 ranged between 8.8 - 11.4 per 100 py in 2009 and 2.3 - 30.8 per 100 py in 2013 (29). Young female PWID may be at greater risk of HCV acquisition than men (34), related to high-risk injecting behaviours, especially in the context of co-existing sexual and injecting relationships (35, 36). Data compiled by the US CDC demonstrated that whilst HCV incidence increased between 2006 - 2012 in all age groups, it was particularly notable in those aged less than 30 years (age 20 – 29: 2010 - 0.75 per 100,000, 2012 - 1.73 per 100,000) (24, 25). This increase coincided with numerous HCV outbreaks among PWID in non-urban communities, frequently associated with prior misuse of prescription opiates (with first use on average 2.0 years before heroin) (24, 37, 38).

Most studies in PWID document similar risk factors in association with HCV seroconversion, namely younger age and shorter time since injecting onset (9, 10, 39-43). Significant risk is associated with shared injecting and ancillary drug preparation equipment (9, 39, 44), with a meta-analysis quantifying risk estimates of several drug equipment sharing behaviours (45). Other baseline factors and behaviours independently associated with seroconversion include injecting frequency, number of injecting partners, type of drug injected, pooling money with other PWID to purchase drugs, unstable housing, HIV co-infection and exchanging sex for money (9, 39, 40, 44, 46). Recent evidence suggests that HCV incidence is reduced in PWID receiving OST (40, 42, 47-49). The factors associated with HCV acquisition in PWID highlight the need for strategies involving integrated multidisciplinary care models to prevent ongoing HCV transmission.

Page 21 of 309 Table 1-1. HCV incidence in general cohorts of adult people who inject drugs, 2010-2014

Cohort Incident Total Study Incidence per 100 Author, year Location Study population number infections follow up period py (95% CI) (n) (n) py

(50) Hope, 2011 Bristol, UK PWID 115 14 NA 2006 38-47

(51) Blome, 2011 Malmo, Sweden PWID 332 186 486 1997-2005 38.9 (33.5, 44.8)

1988-1989 22.0 (12.5, 30.0) (52) Mehta, 2011 Baltimore, US PWID 373 NA NA 2005-2008 7.8 (2.5, 19.0)

(42) Allen, 2012 Scotland PWID 1140 24 NA 2008-2009 10.8-21.9

(53) McDonald, 2012 Glasgow, Scotland PWID 829 173 1674 1993-2007 10.3 (8.9, 12.0)

107 42 109 2002-2004 38.5 (26.8, 50.1) (54) Ruan, 2013 Xichang, China PWID 114 43 142 2006-2008 30.3 (21.2, 39.4)

(40) White, 2014 Sydney, Australia PWID 129 17 215 2008-2011 7.9 (4.9, 12.7)

1996-1999 25.0 (20.2, 30.3) (46) Grebely, 2014 Vancouver, Canada PWID 364 126 1466 2006-2012 3.1 (2.0, 4.8) 6.3 (5.4, 7.3) (47) Nolan, 2014 Vancouver, Canada PWID 1004 184 NA 1996-2012 OST: 0.5 (0.3, 0.8) Abbreviations: OST, opioid substitution therapy; PWID, people who inject drugs; py, person years

Page 22 of 309 Table 1-2. HCV incidence in specific cohorts of adult people who inject drugs, 2010-2014

Cohort Incident Total Study Incidence per 100 py Author, year Location Study population number infections follow period (95% CI) (n) (n) up py

(55) Clatts, 2010 Hanoi, Vietnam PWID <30 years 96 11 47 2005-2006 23.4 (11.7, 41.9)

Vancouver and Prince Indigenous PWID (41) Spittal, 2012 148 45 388 2003-2009 11.6 (8.5, 17.1) George, Canada <30years (56) Sacks-Davis, Melbourne, Australia PWID <30 years 63 19 148 2005-2006 12.8 (7.7, 20.0) 2013 19.8 (19.1, 20.6) San Francisco, (35) Tracy, 2014 PWID <30 years 417 129 650 2000-2012 Men: 17.3 (16.4, 18.3) California Women: 25.4 (24.0, 26.8) San Francisco, (48) Tsui, 2014 PWID <30 years 552 171 680 2000-2013 25.1 (21.6, 29.2) California HIV-positive MSM 620 40 4359 0.9 (0.6, 1.2) (57) Gamage, 2011 Melbourne, Australia 2002-2010 (PWID) (39) (16) (341) (4.7 [2.7, 7.5]) 1998 13.9 (8.2, 22.4) (58) Wandeler, 2012 Switzerland HIV-positive PWID 123 41 733 2011 2.2 (0.6, 10.7) New South Wales, (59) Luciani, 2014 Incarcerated PWID 210 38 269 2005-2009 14.1 (10.0, 19.3) Australia

(60) Marco, 2014 Barcelona, Spain Incarcerated PWID 168 66 NA 1992-2011 6.7

(61) Snow, 2014 Queensland, Australia Incarcerated PWID 77 12 266 1999-2013 5.5 (4.6, 5.6)

Abbreviations: PWID, people who inject drugs; py, person years

Page 23 of 309 The global burden of HIV/HCV co-infection is substantial, with recent estimates suggesting that 1.3–4.4 million HIV-positive individuals are HCV co-infected (62). The natural history of HIV and HCV are significantly impacted by the co-existence of the other virus, with increases in liver-related and all cause morbidity and mortality, even amongst those on combination antiretroviral therapy (cART) (63-66).

In contrast to falling HCV incidence in PWID, an increase in HCV incidence and prevalence has been reported in large cohorts of HIV-positive MSM over the last decade (Table 1-3) (11, 12, 57, 58, 67-73). In a recent meta-analysis examining HCV incidence in HIV-positive MSM who denied ever injecting drugs, estimated annual HCV incidence rates rose from 0.4 per 100 py in 1991 (95% CI 0.2, 0.8) to 1.3 per 100 py in 2012 (95% CI 0.8, 2.4) (12). In the Swiss HIV Cohort Study, HCV incidence increased 18-fold in MSM between 1998 and 2011, while it declined in PWID and remained <1 per 100 py in heterosexuals (58).

HCV incidence is variable and may be stabilising or falling in some settings (74, 75). HCV testing and diagnosis among HIV-positive MSM has increased in line with international guidelines. In Spain, the incidence of HCV diagnosis rose among HIV-positive MSM between 2004 and 2011 (IR 0.8 to 1.1; aIRR 1.5; 95%CI 0.3, 6.8), but HCV incidence fell (IR 1.1 to 0.5; aIRR 0.5; 95%CI 0.1, 2.3) (74). In the UK Collaborative HIV Cohort (UK CHIC) study, while HCV prevalence increased from 7.3% in 2004 to 9.9% in 2011, HCV incidence remained stable (1.0 per 100 py in 2004; 1.4 per 100 py in 2007; 1.1 per 100 py in 2011) (75). Overall, HCV incidence among HIV-positive MSM remains significantly lower than among PWID.

The reported increase in HCV incidence in HIV-positive MSM has been associated with an increase in sexual risk behaviour and recreational drug use (12). While sexual transmission of HCV is uncommon in other populations (76), per-mucosal (sexual) HCV exposure (with blood as the medium) appears to facilitate HCV transmission in HIV-positive MSM (12, 58, 70, 71, 77). Risk factors for HCV acquisition in this population include condom-less traumatic anal intercourse, higher number of sexual partners, group sex, ulcerative sexually transmitted diseases (notably syphilis and lymphogranuloma venereum) and sexual acts that involve trauma and bleeding (12, 58, 70, 71, 77-79). The increase in HCV incidence has occurred in parallel with an increase in the incidence of other sexually transmitted infections and specific behavioural trends in MSM communities, including use of social media sexual networking applications, ‘sero-sorting’ sexual behaviours and the phenomena of ‘chemsex’ (illicit drug use before or during sex, by both injecting and non-injecting routes of administration) (77, 80-83).

While similar risk behaviours are observed in HIV-positive and HIV-negative MSM (84), HCV incidence appears to be markedly lower in HIV-negative MSM. In a systematic review

Page 24 of 309 conducted by Yaphe et al, HCV incidence was 0.6 per 100 py (95% CI 0.5, 0.7) in HIV-positive MSM and 0.2 per 100 py (95% CI 0.1, 0.2) in HIV-negative MSM (85). While uncommon, there have been recent reports of acute HCV infection in HIV-negative MSM receiving HIV pre-exposure prophylaxis (PrEP) who engage in high-risk practices (86). With serosorting of sexual partners by HIV-status and increasing use of PrEP to prevent HIV transmission in HIV- negative MSM, the potential exists for increased sexual risk behaviour and transmission of HCV among MSM populations (77, 86, 87). Current evidence does not support routine HCV screening in HIV-negative MSM, but appropriate testing should occur on a case-by-case basis after potential exposure.

HIV-positive MSM and PWID are not mutually exclusive; HIV-positive MSM who inject drugs are at higher risk of HCV acquisition than HIV-positive MSM who do not inject drugs (11, 57). In a cohort study of HIV-infected MSM, significantly higher HCV incidence was documented among PWID MSM as compared with non-PWID MSM (4.7 per 100 py [95% CI: 2.7, 7.5] vs 0.6 per 100 py [95% CI: 0.4, 0.8]; HR 8.7 [95% CI: 4.6, 16.6]; p <0.001) (57). However, HIV- positive MSM who report injection drug use may exhibit different drug use and sexual behaviours as compared with the non-MSM PWID populations traditionally reported in the HCV literature, and as such different strategies may need to be employed to enhance HCV detection and management.

Page 25 of 309 Table 1-3. HCV incidence in HIV-positive MSM who do not inject drugs, 2010-2015

Cohort Incident Total Study Incidence per 100 py Author, year Location Study population number infections follow period (95% CI) (n) (n) up py 0.5 (0.5, 0.6) 1991: 0.4 (0.2, 0.8) (12) Hagan, 2015 Meta-analysis HIV-positive MSM 13000 497 931000 1984-2012 2010: 1.1 (0.7, 1.6) 2012: 1.3 (0.8, 2.4)

(88) Jin, 2010 Sydney, Australia HIV-positive MSM 129 0 202 2005-2007 0.0 (0.0, 0.2)

(89) Barfod, 2011 Copenhagen, Denmark HIV-positive MSM 871 13 3514 2066-2009 0.4 (0.2, 0.6)

(57) Gamage, 2011 Melbourne, Australia HIV-positive MSM 581 24 4018 2002-2010 0.6 (0.4, 0.8)

0.9 (0.6, 1.3) 1994-2000: 0 (72) Sun, 2012 Taipei, Taiwan HIV-positive MSM 731 28 3026 1994-2010 2001-2005: 0.3 (0.1, 1.0) 2006-2010: 1.2 (0.8, 1.8) 0.4 (0.4, 0.5) (58) Wandeler, 2012 Switzerland HIV-positive MSM 3333 101 23707 1998-2011 1998: 0.2 (0.1, 0.5) 2011: 4.1 (2.6, 6.2)

(90) Witt, 2013 USA HIV-positive MSM 2041 99 20900 1984-2011 0.3 (0.3, 0.4)

0.8 (0.5, 1.3) (73) Nishijima, 2014 Tokyo, Japan HIV-positive MSM 716 17 2146 2005-2012 2005-2006: 0 2011-2012: 2.0 (1.1, 3.7) 0.6 (0.4, 0.9) (74) Sobrino-Vegas, Spain HIV-positive MSM 3621 21 3621 2004-2011 2004-2005: 1.1 (0.2, 3.3) 2014 2010-2011: 0.3 (0.1, 0.8) Abbreviations: MSM, men-who-have-sex-with-men; py, person years Page 26 of 309 Detection of acute HCV infection

Diagnosis

Typically, acute HCV infection is characterised by the appearance of HCV RNA in blood within 2–14 days of exposure, elevation of liver-associated enzymes particularly alanine aminotransferase (ALT), and development of HCV antibodies within 30–60 days of exposure (8). The most accurate case definition of acute HCV infection is detection of HCV RNA in an individual with documented HCV antibody seroconversion. Documentation of seroconversion is difficult in the absence of routine serological testing, but monitoring of at-risk populations may be beneficial, including PWID (91-93) and HIV-positive MSM (91, 94). The detection of HCV RNA with a negative HCV antibody followed by seroconversion suggests very recent infection with probable exposure in the prior 6-8 weeks.

However, while HCV antibodies are usually detectable within 6-12 weeks of infection (95), development may take up to 12 months in immunocompromised patients, including solid-organ transplant recipients (96), haemodialysis patients (97) and HIV-positive individuals (98). In a few cases, HCV antibody formation may not occur. In a cohort of HIV-positive individuals, 5% remained HCV antibody negative at 12 months post diagnosis of acute infection (98). As such, in cases of suspected acute HCV infection and in all immunocompromised individuals, HCV RNA should be included as part of the initial evaluation. Furthermore, individuals who have cleared previous HCV infection remain indefinitely HCV antibody positive, making it an unhelpful marker for diagnosis of reinfection, necessitating HCV RNA testing.

Up to 80% of acute HCV infections are asymptomatic making detection and estimation of duration of infection difficult. Clinical features suggestive of acute infection include significant elevation of ALT or an acute illness manifest by jaundice. However, only 15-30% of those infected develop a symptomatic illness and ALT elevation is non-specific. In the symptomatic population, the average time from HCV exposure to the development of an acute illness is 7 weeks (range 2–12 weeks) (99). Notably, the clinical course of symptomatic acute HCV infection may differ from that of asymptomatic infection with the pronounced inflammatory response increasing spontaneous clearance (16, 17).

While there is no definitive laboratory test to distinguish acute from chronic HCV infection, certain features may be suggestive (Figure 1-1). Acute infection should be suspected if the 4 HCV viral load is low (<10 IU/mL) or fluctuating (>1 log10 IU/mL) (100) or if there is a low signal-to-cut off ratio for HCV antibody with detectable HCV RNA (although the latter result is not routinely available) (101).

Page 27 of 309

Figure 1-1. Viral kinetics in acute HCV infection

Individuals with spontaneous clearance (blue) and viral persistence (plateau with persistence – red; partial viral control with persistence – green).

Reproduced with permission from (102).

Page 28 of 309 Screening strategies and high-risk populations

International guidelines recommend that all PWID (lifetime and recent) should be screened for HCV infection and in the context of ongoing injecting drug use, 6-12 monthly screening with anti-HCV antibody should occur to assess for incident infection (91, 93, 103). Current guidelines also recommend that all newly diagnosed HIV-positive individuals are screened for HCV antibody (91, 94). If negative, HIV-positive MSM at risk of acquiring HCV should be screened at 6-monthly intervals with ALT and annually with anti-HCV antibody (91, 94). After potential exposure (including injection drug use and/or high risk sexual behaviour) or diagnosis of a sexually transmitted infection, HCV screening should be also undertaken and repeated 3 months later (94). Screening protocols for acute HCV in specific high-risk populations, including young PWID (35, 104, 105), incarcerated PWID (33, 106) and HIV-positive MSM (58, 107), should be considered to enhance HCV diagnosis, prevention and surveillance.

Management of acute and recent HCV infection

Natural history and spontaneous clearance The management of acute HCV infection is inextricably linked to the natural history of acute and early chronic HCV infection with early treatment initiation balanced against the potential for spontaneous clearance. In contrast to chronic HCV infection, acute infection displays a dynamic course with marked fluctuation in HCV RNA and ALT levels. From exposure to seroconversion, three phases have been described: the “pre-ramp up” phase (2-14 days) with intermittent low level viraemia (potentially below the level of detection), the “ramp up” phase (8-10 days) with an exponential rise in HCV RNA and the “plateau” phase (45-68 days) in which HCV RNA levels stabilise (108). This is followed by either viral persistence (75%) or spontaneous clearance (25%) (16, 17) (Figure 1-1).

Most individuals spontaneously clear HCV in the first six (67-86%) or 12 months (83–95%) months following infection (17, 104, 109-111). Spontaneous clearance is associated with female gender (16, 17, 102, 104), younger age, HCV genotype 1 (17, 112), symptomatic acute hepatitis at presentation (16, 112), host genetic factors (17), higher HCV RNA at diagnosis and higher infectious inoculum (113). Single-nucleotide polymorphisms (SNPs) near the interleukin-28 (IL28B) gene region, which encodes the interferon-λ4 protein (IFN-L4), have been identified that strongly predict clearance, both spontaneous and in response to interferon- based therapy (17). While it is still uncertain which specific immunological characteristics during the acute phase of HCV infection predict clearance or persistence, the failure of a sustained T-cell response, particularly CD4+ T helper activity, appears to predict persistence

Page 29 of 309 (114, 115). In a cohort of HIV-positive men with acute HCV infection, the best clinical predictors of spontaneous clearance were maximum log10 fall in HCV viral load within 100 days, peak bilirubin (≥40 mmol/L), peak ALT (≥1000 IU/L) and baseline CD4 count (≥650×106/L) (112). As expected, immunocompromised hosts have a lower chance of spontaneous clearance (≤20%) (98, 112, 116-120), which is of particular importance given the high incidence of HIV co-infection in specific populations of PWID and MSM.

Timing of HCV treatment initiation Given the potential for spontaneous clearance, the decision regarding timing of treatment initiation is important. Deterding et al performed a randomised non-inferiority trial in which adults with symptomatic acute HCV mono-infection (n=107) were allocated (1:1) to receive immediate PEG-IFN alfa-2b or delayed (12 weeks after randomisation) PEG-IFN alfa-2b and ribavirin for 24 weeks (121). All asymptomatic subjects received immediate PEG-IFN alfa-2b. By ITT analysis of the symptomatic individuals, 67% (37/55) in the immediate treatment arm and 54% (28/52) in the delayed treatment arm achieved SVR (difference 13.7%, 95% CI: 4.6, 32.0; p=0.071). In the asymptomatic subjects, 72% (18/25) demonstrated SVR. Of note, loss to follow up was significant, particularly in the delayed treatment arm, likely contributing to the lower SVR in this arm (42% [22/52] delayed treatment; 25% [20/80] immediate treatment, symptomatic or asymptomatic; p=0.037). For those who completed follow up, SVR was 90% (37/41) and 93% (28/30) in the immediate and delayed symptomatic treatment arms, respectively, and 95% (20/21) in the asymptomatic arm. In a meta-analysis by Corey et al, the estimated SVR among patients initiating treatment within 12, 12–24 and more than 24 weeks from time of diagnosis was 83%, 67% and 63%, respectively (122). This data suggests that delaying treatment for 12 weeks following initial diagnosis of acute infection provides an opportunity to assess for spontaneous clearance without compromising outcome, while a more prolonged delay is not beneficial and may risk losing individuals to follow-up.

Interferon-based treatment of acute HCV mono-infection Global clinical practice of acute HCV mono-infection is not standardised with significant uncertainty regarding the optimal regimen and treatment duration, particularly as the therapeutic landscape changes with the advent of DAA therapy for chronic HCV. Previous international guidelines had been in agreement regarding administration of 16-24 weeks of PEG-IFN monotherapy. In 2015, this position was still supported by European guidelines (103). However, despite the lack of direct evidence, amendments to the 2015 US guidelines supported “the same regimens recommended for chronic HCV infection … owing to high efficacy and

Page 30 of 309 safety” (class IIa, level C) with PEG-IFN with or without ribavirin for 16-24 weeks listed as an alternative regimen (class II, level A) (91).

Early clinical trials evaluating standard or PEG-IFN monotherapy for acute HCV mono- infection demonstrated promising results (largely in symptomatic patients) with the proportion achieving SVR24 ranging between 75-98% (123-126). The first large prospective study evaluated 44 patients (mean duration of infection prior to treatment initiation: 89 days; genotype 1, 61%) who received daily standard interferon alfa-2b for 4 weeks, followed by thrice weekly interferon for a further 20 weeks with a resultant SVR24 of 98% (123). Santantonio et al demonstrated high efficacy (SVR24 94%) with 24 weeks of PEG-IFN alfa-2b in 16 patients with detectable HCV RNA 12 weeks following the onset of acute HCV infection (125). In a larger German prospective trial, 89 patients (median duration of infection: 76 days) received PEG-IFN alfa-2b for 24 weeks with an SVR24 of 71% in the ITT population (per-protocol SVR24 89%) (127). A meta-analysis by Corey et al of 22 studies (including 1075 patients) revealed an overall SVR of 78% for acute HCV treated with standard or PEG-IFN monotherapy for a mean treatment duration of 19.7 weeks (standard deviation, 12.5 weeks) (122).

More recently, Santantonio et al evaluated the efficacy and safety of a 24 week course of PEG- IFN alfa-2b (group A) versus a 12-week course of PEG-IFN alfa-2b alone (group B) or with ribavirin (group C) in an open label, randomized multicentre trial involving 130 patients with acute HCV mono-infection who did not demonstrate spontaneous clearance after 12 weeks of observation (128). The study protocol excluded PWID. By ITT, the overall SVR24 was 72%; specifically, SVR24 was 71% (31/44), 72% (31/43) and 72% (31/43) in groups A, B and C, respectively (p=0.898). By per-protocol analysis, SVR24 rose to 82% in each group. On multivariate analysis, a rapid virological response (RVR, undetectable HCV RNA at week 4 of treatment) was associated with SVR, consistent with the evidence available in chronic HCV infection. No other assessed baseline factor (age, gender, HCV genotype, treatment arm) predicted SVR.

The addition of ribavirin to PEG-IFN is of uncertain benefit in acute HCV mono-infection. The recent study by Santantonio et al (128) and others (129) demonstrated no additional advantage. In the Australian Trial in Acute Hepatitis C (ATAHC) trial, the addition of ribavirin in the HIV co-infected population improved viral kinetics as compared with PEG-IFN monotherapy in the HCV mono-infected population (130).

Page 31 of 309 Interferon-based treatment of recent HCV infection in PWID While the majority of HCV transmission in the developed world occurs in the setting of injecting drug use, there are limited data on treatment of acute HCV infection in PWID. In a small Swiss study, 22 patients with acute HCV were assessed for treatment and 14 commenced PEG-IFN alfa-2b for 24 weeks (131). By ITT, the SVR24 was 57%. However, adherence was poor with only 8 patients completing more than 80% of the scheduled treatment course (SVR by per-protocol analysis, 88% [7/8]). De Rosa et al offered 12 weeks of directly observed therapy with PEG-IFN alfa-2b with a much improved SVR 24 of 74% (17/23) (132). Adherence was excellent with 96% (22/23) completing more than 80% of the scheduled treatment course.

The largest trial to date examining treatment of acute and early chronic HCV infection involving PWID was ATAHC, a multicentre prospective cohort study of the natural history and outcomes following treatment with PEG-IFN alfa-2a with or without ribavirin (133). Between 2004 and 2008, 167 subjects were enrolled, of whom 111 (76%) with detectable HCV RNA at screening elected to commence treatment. Of the treated population, 57% had HCV genotype 1, 85% had acquired HCV via injection drug use and the median estimated duration of infection was 25 weeks. HIV co-infection was documented in 33%, the majority of whom had acquired HCV via sexual (per-mucosal) transmission. In the cohort of 74 HCV mono-infected individuals who commenced PEG-IFN monotherapy, SVR24 by ITT and per protocol analyses were 55% and 72%, respectively, with 77% (57/74) receiving at least 80% of the scheduled treatment course. While the proportion achieving SVR was lower in those who had a history of injection drug use (history of injection drug use, SVR 24 48% vs no injection drug use, SVR 24 91%; p=0.03), after adjusted analysis, the only pre-treatment factors associated with treatment failure were poor social functioning and a history of drug dependency treatment (133).

Collectively, these data demonstrate that PWID with recent HCV infection can be treated successfully, but that strategies to optimize adherence in socially marginalized populations are required to improve outcomes.

Interferon-based treatment of recent HCV infection in HIV co-infection HIV co-infection does not appear to compromise efficacy with interferon-based therapy in acute HCV infection, in contrast to chronic HCV infection (134, 135). In the ATAHC trial, 35 patients co-infected with HIV and HCV commenced 24 weeks of PEG-IFN and ribavirin with SVR24 by ITT and per protocol analyses of 74% and 75%, respectively (133). The higher SVR among HIV-positive compared with HIV-negative individuals may relate to socio-demographic differences between these groups with the majority of HIV-positive individuals reporting higher levels of education and employment, more stable housing and greater social support than the

Page 32 of 309 HIV-negative patients, facilitating better adherence. Acknowledging the predominance of sexual (per-mucosal) transmission (60%), injection drug use remained an important mode of acquisition in the co-infected cohort (37%), with 54% reporting prior or current injecting drug use.

Boescke et al demonstrated similar results with administration of either PEG-IFN alfa-2a monotherapy or PEG-IFN and ribavirin for 24-48 weeks at the investigators discretion (treatment duration 24 weeks: PEG-IFN 83%, PEG-IFN/ ribavirin 70%) (136). The overall SVR was 65%, with no apparent additional benefit of ribavirin (PEG-IFN SVR 69%, PEG-IFN/ ribavirin SVR 63%). The European AIDS Treatment Network (NEAT) reviewed nine co- infected cohorts, including 170 HIV-positive individuals who received IFN-based treatment for acute HCV infection (predominantly genotype 1) and demonstrated a combined SVR24 of 60- 80% (94). Their subsequent recommendations involved administration of 24-48 weeks of PEG- IFN and ribavirin, with treatment duration based on presence or absence of RVR.

Duration of interferon-based treatment in recent HCV infection The enhanced outcomes in recent HCV infection, translating as superior SVR in comparison with that achieved in chronic HCV infection, allow interferon-based therapy to be simplified, and specifically be administered for a shorter duration. Shorter treatment durations result in fewer adverse events, better quality of life, less frequent dose reductions and increased likelihood of optimal adherence (137). Studies have demonstrated the efficacy of short course treatment for four (SVR24 87%) and 12 weeks (SVR24 72–74%) in predominantly symptomatic recent HCV infection (124, 128, 137, 138). Administration of interferon in acute HCV infection may play a unique role in enhancing outcome due to differences in expression of interferon-stimulated genes (139). However, the development of direct-acting antiviral regimens for chronic HCV infection with extremely high efficacy and shortened duration has markedly reduced the relevance of enhanced interferon-based treatment outcomes in acute HCV infection.

Evidence gaps related to the treatment of acute and recent HCV infection As the HCV therapeutic landscape evolves with the availability of DAA therapy, the optimal management of recent (acute or early chronic) HCV infection is uncertain. Much of what is known about the timing of treatment initiation, regimen choice and duration of therapy in acute HCV infection comes from small observational studies and randomized controlled trials using interferon-based therapy in selected populations. Excellent results with DAA therapy in chronic

Page 33 of 309 HCV infection have cast some doubt on any “efficacy advantage” of early treatment in acute HCV infection (140). While administration of interferon in acute HCV infection appeared to play a unique role in enhancing outcome due to differences in expression of interferon- stimulated genes (139), the impact of host genetics and immune response on treatment outcome with DAA therapy in recent HCV infection is unknown. The impact of clinical, virological and immunological factors on DAA efficacy in acute HCV infection, such as HIV infection, baseline HCV RNA, mode of HCV transmission, clinical presentation and duration of infection, remain to be determined. As interferon-free DAA therapy is established as the standard-of-care for chronic HCV infection (91, 103, 140), its role and activity in recent HCV infection requires evaluation.

Page 34 of 309 Overview of Protease, Polymerase and Assembly Inhibitors for the Treatment of HCV Following the discovery of HCV in 1989 (141), HCV had been hampered by the absence of a robust cell culture system for HCV infection. However, a number of virological breakthroughs have led to a new age in rational drug design for HCV infection. These include the identification of an infectious HCV sequence in chimpanzees (142), replication of sub-genomic HCV RNA in a human hepatoma cell lines (143), HCV replication in SCID mice with chimeric human livers (144) and the production of infectious HCV in tissue culture from a cloned viral genome (145). These advances in molecular biology led to a paradigm shift from traditional high-throughput drug screening towards rational, structure-based drug design for the identification of novel antiviral agents for the treatment of HCV infection (140, 146).

As the HCV viral life cycle has been more fully elucidated, rational drug design and screening of large compound libraries have been used to identify small molecule inhibitors of various HCV proteins involved in HCV replication. The three most clinically important proteins include (1) the non-structural (NS) 3/4A protease, which is involved in post-translation processing of HCV polyproteins and impairs the production of endogenous interferon by infected cells; (2) the NS5B RNA-dependent RNA polymerase (RdRp), which is required for copying the HCV RNA genome and is essential for viral replication; and (3) the NS5A protein, which is involved in the formation of the replication complex and viral assembly.

Employing drug development strategies similar to those used for developing antiretroviral drugs for HIV, numerous inhibitors of these three viral targets are approved for use (Table 1-4) or in clinical development (Table 1-5, Supplementary Appendix Table 1-1) (140). The four classes of HCV DAAs approved for clinical use are defined by their mechanism of action and therapeutic target: 1. NS3/4A protease inhibitors, 2. nucleotide analogue NS5B RdRp inhibitors, 3. non-nucleoside RdRp inhibitors, 4. NS5A inhibitors.

Page 35 of 309 Table 1-4. Direct-acting antiviral agents approved for chronic HCV infection

Year of Molecular Compound & HCV Trade name FDA Molecular formula weight class GT approval (g/mol) NS3/4A Protease Inhibitors First-generation, first wave

Boceprevir Victrelis® 2011 1 C27H45N5O5 519.68

Telaprevir Incivek® 2011 1 C36H53N7O6 679.85 First generation, second wave

Simeprevir Olysio® 2013 1 C38H47N5O7S2 749.94 5 Sunvepra® 2014 1 C35H46ClN5O9S 748.29 1 Viekira Pak® 2014 1, 4 C40H43N7O7S•2H2O 801.91 6 2014 1 C38H55N5O9S 757.94 Second generation 2 Zepatier® 2016 1, 4 C38H52N6O10S 784.92 NS5B RNA-dependent RNA Polymerase Inhibitors Nucleotide

Sofosbuvir Sovaldi® 2013 1-6 C22H29FN3O9P 529.45 Non-nucleoside 1 Viekira Pak® 2014 1 C26H26N3O5S•Na•H2O 533.57 NS5A Inhibitors First generation, first wave

Daclatasvir Daklinza® 2015 1-6 C40H50N8O6 738.88 3 Harvoni® 2014 1 C49H54F2N8O6 889.00 1 Viekira Pak® 2014 1, 4 C50H67N7O8•4.5H2O 975.20 First generation, second wave 2 Zepatier® 2016 1, 4 C49H55N9O7 882.02 4 Epclusa® 2016 1-6 C49H54N8O8 883.01 1 Viekira Pak: Co-packaged ombitasvir/paritaprevir/ co-formulated fixed dose combination and dasabuvir. Ritonavir is not active against HCV. Ritonavir is a potent CYP3A inhibitor that increases peak and trough plasma drug concentrations of paritaprevir and overall drug exposure. 2 Zepatier: Grazoprevir/elbasvir 100mf/50mg co-formulated fixed dose combination 3 Harvoni: Sofosbuvir/ledipasvir 400mg/100 mg co-formulated fixed dose combination 4 Epclusa: Sofosbuvir/velpatasvir 400mg/100mg co-formulated fixed dose combination 5 Asunaprevir: Approved in Asia and Middle East 6 Vaniprevir: Approved in Japan

Chemical structure of approved DAAs depicted in Supplementary Appendix, Figures 1-1 – 1-14. Abbreviations: GT, genotype

Page 36 of 309 Table 1-5. Investigational Phase II and III direct-acting antiviral agents for HCV infection

Molecular Compound Development HCV Formulation Molecular formula weight & class stage GT (g/mol) NS3/4A Protease Inhibitors Second generation Phase III Oral 1-6 C H F N O S 838.87 (ABT-493) 38 46 4 6 9 Phase III Oral 1-6 C H F N O S 868.94 (GS-9857) 40 52 4 6 9 Vedroprevir Phase II Oral 1 C H C N O S 910.52 (GS-9451) 45 60 l 7 9 NS5B RNA-dependent RNA Polymerase Inhibitors Nucleotide analogues AL-335 Phase II Oral 1-6 Undisclosed Undisclosed MK-3682 Phase II Oral 1-6 Undisclosed Undisclosed NS5A Inhibitors First generation, second wave Phase II/III Oral 1-6 C H N O 1001.26 (ACH-3102) 60 72 8 6 Phase III Oral 1-6 C H N O 762.90 (PPI-668) 42 50 8 6 Second generation Phase III Oral 1-6 C H F N O 1113.18 (ABT-530) 57 65 5 10 8 MK-8408 Phase II Oral 1-6 Undisclosed Undisclosed MicroRNA-122 Inhibitors

Miravirsen Phase IIa Subcutaneous 1-6 C151H185N49O83P14S14 4896.87 RG-101 Phase IIa Subcutaneous 1-6 Undisclosed Undisclosed Cyclophilin Inhibitors Alisporivir Phase IIb/III Oral 1-6 C H N O 1216.64 (Debio-025) 63 113 11 12

*Investigational DAAs in late stages of development which have ceased ongoing study due to suboptimal efficacy or safety concerns are not included.

Examples of such agents include (by class): NS3/4a protease inhibitors – (ITMN-191; PubChem CID: 71301228), (PubChem CID: 71300931), (SCH-900518; PubChem CID: 11857239), (ACH-0141625; PubChem CID: 53362096); NS5B nucleotide/non- nucleoside inhibitors – (BMS-791325; PubChem CID: 72722244); BMS-986094 (INX-189; PubChem CID: 46700744), (PubChem CID: 56948249), (PF-868554; PubChem CID: 54708673), (PubChem CID: 16122663), nesbuvir (HCV-796; PubChem CID: 11561383), radalbuvir (GS-9669; PubChem CID: 53259022), (ANA-598; PubChem CID: 45136829), tegobuvir (GS-9190; PubChem CID: 23649154); NS5A inhibitor: (IDX-719; PubChem CID: 53302707).

Compiled from: (147-163)

Page 37 of 309 Mechanism of drug action HCV is a positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Its 9600 nucleotide genome encodes a 3000 amino acid precursor polyprotein. This is further cleaved into three structural (core, E1, and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (164, 165). Some of these HCV proteins are the key targets for HCV therapy and vaccine design.

The initial stages of the HCV replication cycle include binding to cell surface proteins, cell entry mediated by endocytosis and fusion and uncoating inside the target cell (166). Following decapsidation of viral nucleocapsids in the cell, positive-strand HCV RNA is liberated, serving as HCV messenger RNA (mRNA) for translation, thereby forming the HCV polyproteins. Post- translational processing of HCV proteins occurs mainly through the actions of two cellular peptidases (host signal peptidase and signal peptide peptidases) and two viral proteases, NS2 and NS3/4A leading to cleavage of downstream NS proteins through NS3/4A cofactors. Subsequently, HCV replication occurs via the formation of a replication complex, which includes the NS5B RNA-dependent RNA polymerase. Unwinding of double strand HCV RNA is then facilitated through the NS3 helicase-NTPase and positive-strand HCV RNA is used for the generation of negative-strand HCV RNA intermediates. This negative-strand HCV RNA is then subsequently used as a template for the production of numerous positive-strand HCV mRNA copies which are subsequently used for polyprotein translation, synthesis of new intermediates of replication or packaging into new virus particles. The virus polyproteins and positive-strand HCV RNA are assembled, packaged and transported to the cell surface, with ongoing maturation of glycoproteins during this time. Vesicle fusion with the cell surface then results in the release of infectious mature virions from the cell.

HCV NS3/4A protease inhibitors NS3/4A drugs inhibit the NS3/4A serine protease, thereby blocking the synthesis of pivotal proteins involved in post-translational processing and replication of HCV. NS3 has multiple functions including a chymotrypsin-like serine protease domain at its N-terminus and a helicase domain at the C-terminus. NS4A is a cofactor for NS3 activity. The NS3/4A serine protease cleaves the viral polyprotein at four junctions (NS/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B) with considerable peptide sequence similarity. The crystal structure of NS3/4A complex has been determined (167-169) and the catalytic domain is formed by a triad consisting of His57, Asp81 and Ser139 (170-172). Proper folding of the C-terminal protease domain also requires the tetrahedral coordinated binding of a zinc ion. NS3/4A protease inhibitors disrupt HCV by blocking the NS3 catalytic site or the NS3/NS4A interaction. In addition to its role in

Page 38 of 309 viral processing, the NS3/NS4A protease blocks TRIF-mediated Toll-like receptor signalling and Cardif-mediated retinoic acid–inducible gene 1 (RIG-1) signalling; this results in impaired induction of and the innate host response. .

HCV NS5B RNA-dependent RNA polymerase inhibitors The NS5B RNA-dependent RNA polymerase (RdRp) is the key enzyme mediating viral RNA synthesis (173). The structure of the catalytic core resembles a right hand with a classical “fingers, thumb and palm” structure of RNA polymerases (174-176). NS5B contains a C- terminal α-helical transmembrane domain which anchors the protein to the cytosolic side of the endoplasmic reticulum (ER) and a C-terminal RNA-dependent RNA polymerase domain. The highly conserved “palm” of the polymerase forms the active site of NS5B, while the “finger and thumb” domains interact to modulate interactions with the growing RNA chain. The fingertip subdomains, formed by two loops, interact specifically with the thumb domain, which forms a catalytic tunnel incorporating the RNA template, the growing RNA strand and nucleotide substrates required for elongation of the nascent RNA strand. An open-conformation of the NS5B polymerase can be adopted, whereby the fingers and thumbs contact is disrupted, with wider access to the catalytic site.

Two classes of NS5B RdRp inhibitor have been developed: nucleotide analogues (i.e., sofosbuvir), which are mimics of the natural substrates of the polymerase and as such, target the active catalytic site of the enzyme, resulting in chain termination and irreversible cessation of RNA synthesis; and non-nucleoside inhibitors (i.e., dasabuvir), which bind to allosteric sites and inhibit conformational change, but only reversibly block RNA synthesis.

Nucleotide analogue inhibitors of HCV RdRp are derivatives of ribonucleotides and compete with natural ribonucleotide substrates for binding to the active site of the polymerase, requiring higher intracellular concentrations than other HCV antiviral drugs to be effective. These drugs are typically delivered as mono-phosphorylated prodrugs carrying chemical modifications to enhance uptake and bioavailability (177). Prior to the discovery of sofosbuvir, a variety of nucleoside analogues had been examined (e.g. mericitabine), but these exhibited relatively low potency related in part to slow initial mono-phosphorylation by nucleoside kinases (178-180). The design of sofosbuvir avoids this by building the first phosphate group into the structure of the drug during synthesis; additional groups are attached to the phosphorus to temporarily mask the two negative charges of the phosphate group to facilitate drug entry into the HCV infected cell (181). Subsequent intracellular di- and tri-phosphorylation of sofosbuvir occurs rapidly via nucleotidyl and nucleoside diphosphate kinases to form the pharmacologically-active uridine analogue triphosphate (GS-461203), which is incorporated into HCV RNA by the NS5B

Page 39 of 309 polymerase and acts as a chain terminator, irreversibly terminating RNA synthesis. Owing to the highly conserved active site, as compared to the allosteric binding sites, nucleotide inhibitors are generally effective against a broader range of viral genotypes and have a higher barrier to resistance than non-nucleoside inhibitors.

The four allosteric sites that act as targets for non-nucleoside inhibitors of RdRp are thumb domains 1 and 2 and palm domains 1 and 2 of the polymerase. In general, NNPIs are less potent, more genotype specific (all non-nucleoside inhibitors in clinical development have been optimized for genotype 1), have a low to moderate barrier to resistance, and have variable toxicity profiles.

HCV NS5A assembly inhibitors The NS5A is a 447 amino acid, zinc-binding phosphoprotein comprised of three domains separated by two linker regions (182, 183). Domain I contains the Zn2+- and RNA-binding motifs and has been crystallized as a dimer (182). It has been suggested that this dimer oligomerizes to form a protective replication compartment that tethers the HCV RNA to intracellular membranes (184). Domain II binds to numerous host proteins and some of these interactions have been linked to RNA replication (185, 186). Domain III is important for virus assembly but is not required for replication (185). Structurally, the amino-terminus of NS5A comprises the amphipathic α-helix, which is responsible for anchoring to the ER and ER- derived membranes, including lipid droplets (LDs) (183, 187).

The NS5A protein is essential for several steps in viral replication, virion assembly and release. Like virtually all plus-strand RNA viruses, NS5A has the hallmark feature of forming a membrane-associated replication complex composed of virus proteins, replicating RNA and altered cellular membranes.

The exact mechanism of action of HCV NS5A inhibitors, exemplified by , remains unclear. Available evidence suggests that NS5A inhibitors have multiple effects, including inhibition of viral RNA synthesis and blockade of virion assembly and secretion, which contribute to their potency. A study of nine HCV-infected individuals who were administered a single dose of daclatasvir (10 or 100 mg) demonstrated a triphasic decline in serum HCV RNA (188). The triphasic model predicted two phases of viral decline in the first 48 hours of daclatasvir administration followed by the third phase after 48 hours. An initial, rapid two-log10 decline in HCV RNA over the first 6 hours (HCV RNA decline from baseline at 2, 4, and 6 hours post dose: 0.27, 1.20 and 1.95 log10 IU/mL) was followed by a slower phase of decline (6 - 48 hours). The successive phases of decline were deemed to represent the effect of daclatasvir

Page 40 of 309 on different stages of the viral lifecycle, namely rapid initial blockade of virion assembly and secretion (first phase) followed by inhibition of RNA synthesis (second phase). The third phase reflected long-term viral decline, driven by the loss rate of HCV infected cells. These clinical findings suggest that NS5A inhibitors have at least two mechanisms of action that affect both viral replication and viral assembly/release. Subsequently, Sun et al (189) developed a model for NS5A inhibitor action, in which NS5A proteins interact with each other, a single bound inhibitor disrupts the function of an NS5A oligomer and impairs the formation of the replication complex or the function of NS5A within the replication complex and amplifies the inhibitory effect. Of significant interest, Sun et al. demonstrated that while daclatasvir and an NS5A inhibitor analogue (Syn-395) were inactive against certain NS5A resistant variants, in combination the pair enhanced daclatasvir potency by >1,000-fold (189). This interaction between the pair of compounds suggests that the NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, desensitising resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity provides additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle (189).

Page 41 of 309 Mode of drug administration and dosage Adults The recommended doses for individual or fixed-dose combination HCV DAA drugs are listed in Table 1-6, Table 1-7 and Table 1-8. All approved HCV DAAs are formulated for oral administration only. HCV DAAs should not be administered as monotherapy; HCV DAAs should always be co-administered with one or more agents from another class of DAAs, PEG- IFN and/or ribavirin.

Newborn infants and children The safety, efficacy and pharmacokinetic profiles of DAAs have not been established in pediatric patients hence there are no recommendations for use in this population. However, data are emerging with the first report of successful treatment of HCV genotype 1 infection in adolescents (age 12-17 years) with sofosbuvir/ledipasvir in 2016 (190); a study in children aged 3 – 12 years is ongoing.

Pregnant and lactating mothers There are no adequate and well-controlled studies of HCV DAAs in pregnant women. Those drugs that require co-administration with PEG-IFN α and/or ribavirin are classed as Pregnancy Category X. Animal studies have demonstrated that ribavirin causes birth defects and/or fetal deaths while PEG-IFNα is abortifacient.

While no data with daclatasvir exists in pregnant women, administration is not recommended (US FDA pregnancy category: Not assigned; AU TGA pregnancy category: B3) (191). In animal reproduction studies in rats and rabbits, embryo-fetal toxicity was observed in maternally toxic doses that produced exposures of 33 and 98 times the human exposure, respectively, at the recommended human dose of 60 mg.

Other HCV DAA drugs are classed as Pregnancy Category B (including sofosbuvir, sofosbuvir/ledipasvir, ombitasvir/paritaprevir/ritonavir, dasabuvir). They should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus.

Given lack of data, nursing mothers are advised to discontinue breastfeeding prior to commencement of HCV drug therapy.

Page 42 of 309 Table 1-6. HCV NS3/4A protease inhibitors: Administration and dosage in adults

Drug name Formulation Routine adult dosage Altered dosages Single drug class NS3/4A protease inhibitors Impaired renal function No dose adjustment 200 mg 800 mg three times daily Impaired hepatic function Caution in moderate and severe hepatic impairment; no data capsule Pregnancy and lactating women Pregnancy Category X, if with PEG-IFN and RBV 750 mg three times a No dose adjustment in mild renal impairment Impaired renal function day with food (≥20g fat) No data in HCV-infected adults with CrCl <50mL/min Telaprevir 375 mg tablet OR Impaired hepatic function Not recommended in moderate and severe hepatic impairment 1125 mg twice daily Pregnancy and lactating women Pregnancy Category X, if with PEG-IFN alfa and RBV with food (≥20g fat) Impaired renal function No dose adjustment 150 mg 150 mg daily with food Impaired hepatic function Not recommended in moderate and severe hepatic impairment capsule Pregnancy and lactating women Pregnancy Category X, if with PEG-IFN alfa and RBV Impaired renal function Dose reduce (100 mg daily) if CrCl <30mL/min 100 mg Asunaprevir 100 mg twice daily Impaired hepatic function Contraindicated in moderate and severe hepatic impairment capsule Pregnancy and lactating women Not recommended; contraindicated with daclatasvir Multiple drug classes in fixed dose combination NS3/4A protease inhibitor + NS5A inhibitor No dose adjustment in mild - severe renal impairment Impaired renal function Ombitasvir + Preliminary data in ESRD/haemodialysis: No dose adjustment 25mg/150mg/100mg paritaprevir 12.5/75/50mg No dose adjustment in mild hepatic impairment daily Impaired hepatic function + tablet Contraindicated in moderate and severe hepatic impairment (Two tablets daily) ritonavir Pregnancy Category B Pregnancy and lactating women Pregnancy Category X, if co-administered with RBV Impaired renal function Suitable for use in ESRD/haemodialysis Grazoprevir 100mg/50mg 100mg/50mg daily No dose adjustment in mild hepatic impairment + tablet (One tablet daily) Impaired hepatic function Preliminary data: Moderate - 50mg/50mg daily elbasvir No data in severe (Child Pugh Class C) hepatic impairment Pregnancy and lactating women No data 1 Degree of hepatic impairment: Mild refers to Child Pugh Class A, moderate refers to Child Pugh Class B and severe refers to Child Pugh Class C.

Page 43 of 309 Table 1-7. HCV NS5B polymerase inhibitors: Administration and dosage in adults

Drug name Formulation Routine adult dosage Altered dosages Single drug class NS5B polymerase inhibitors No dose adjustment for GFR >30 mL/minute Impaired renal function No recommendation for GFR <30mL/min, haemodialysis 400 mg Sofosbuvir 400 mg daily Impaired hepatic function No dose adjustment tablet Pregnancy Category B Pregnancy and lactating women Pregnancy Category X, if with PEG-IFN alfa or RBV No dose adjustment in mild - severe renal impairment Impaired renal function No data in ESRD/haemodialysis 250 mg No dose adjustment in mild hepatic impairment Dasabuvir 250 mg twice daily Impaired hepatic function tablet Contraindicated in moderate and severe hepatic impairment Pregnancy Category B Pregnancy and lactating women Pregnancy Category X, if co-administered with RBV Multiple drug classes in fixed dose combination NS5B polymerase inhibitor + NS5A inhibitor No dose adjustment for GFR >30 mL/minute Impaired renal function No dose recommendation GFR <30mL/min or haemodialysis Sofosbuvir + 400/90mg 400mg/90mg daily Impaired hepatic function No dose adjustment ledipasvir tablet (One tablet daily) Pregnancy and lactating women Pregnancy Category B The elderly No dose adjustment No dose adjustment for GFR >30 mL/minute Impaired renal function No data for with GFR <30mL/minute Sofosbuvir + 400/100mg 400mg/100mg daily No dose adjustment in mild – severe hepatic impairment. velpatasvir tablet (One table daily) Impaired hepatic function Administer with ribavirin in moderate to severe impairment Pregnancy and lactating women No data 1 Degree of hepatic impairment: Mild refers to Child Pugh Class A, moderate refers to Child Pugh Class B and severe refers to Child Pugh Class C.

Page 44 of 309 Table 1-8. HCV NS5A inhibitors: Administration and dosage in adults

Routine adult Drug name Formulation Altered dosages dosage Single drug class NS5A inhibitor Impaired renal function No dose adjustment Impaired hepatic function No dose adjustment 30 mg tablet Pregnancy and lactating women Contraindicated Daclatasvir 60 mg daily 60 mg tablet Co-administration with: Strong CYP3A inhibitors* 30 mg daily Moderate CYP3A inducers* 90 mg daily Multiple drug classes in fixed dose combination NS3/4A protease inhibitor + NS5A inhibitor No dose adjustment in mild - severe renal impairment Impaired renal function Preliminary data in ESRD/haemodialysis: No dose adjustment Ombitasvir + 25mg/150mg/100mg 12.5/75/50mg No dose adjustment in mild hepatic impairment paritaprevir + daily Impaired hepatic function tablet Contraindicated in moderate and severe hepatic impairment ritonavir (Two tablets daily) Pregnancy Category B Pregnancy and lactating women Pregnancy Category X, if co-administered with RBV Impaired renal function No dose adjustment; suitable for use in ESRD/haemodialysis No dose adjustment in mild hepatic impairment Grazoprevir + 100mg/50mg 100mg/50mg daily Impaired hepatic function Preliminary data: Moderate - 50mg/50mg daily elbasvir tablet (One tablet daily) No data in severe hepatic impairment Pregnancy and lactating women No data NS5B polymerase inhibitor + NS5A inhibitor No dose adjustment for GFR >30 mL/minute Impaired renal function Sofosbuvir + 400mg/90mg 400mg/90mg daily No recommendation for GFR <30mL/min or haemodialysis ledipasvir tablet (One tablet daily) Impaired hepatic function No dose adjustment Pregnancy and lactating women Pregnancy Category B No dose adjustment for GFR >50 mL/minute Impaired renal function Sofosbuvir + 400mg/100mg 400mg/100mg daily No data for GFR <50mL/minute velpatasvir tablet (One table daily) Impaired hepatic function No dose adjustment in mild or moderate hepatic impairment Pregnancy and lactating women No data *Strong CYP3A inhibitors (i.e., ritonavir-boosted atazanavir, some azoles, clarithromycin); moderate CYP3A inducers (i.e., efavirenz, dexamethasone, nafcillin)

Page 45 of 309 Adverse reactions and toxicity Pegylated interferon and ribavirin Contemporary DAA regimens are largely interferon-free and increasingly, do not require ribavirin, except in specific populations (for example, cirrhosis and retreatment following DAA failure). However, it is worth recapping the adverse effects of PEG-IFN and ribavirin, as this provides a useful comparator.

Adverse events affect virtually all patients who receive treatment with PEG-IFN and ribavirin combination therapy (Figure 1.2). The most common side effects attributed to PEG-IFN include myalgia (40-55%) and fatigue (65%) (192-196). Flu-like symptoms (65%) and cytopenias (specifically neutropenia; 30% and thrombocytopenia; 5%) are also commonly observed. Interferon can also lead to neuropsychiatric side effects in those with and without pre- existing psychiatric disease, including depression (20-30%), anxiety/irritability (35-45%), sleep disturbance (30-40%) and poor concentration (10-15%) (195-199). Other common side effects include anorexia (25-30%), nausea (25-40%), rash (10-25%), diarrhoea (20%), arthralgia (25%), headaches (40-60%) and dizziness (15-20%).

With respect to ribavirin, the most commonly reported adverse event is haemolysis, which may lead to clinically significant anemia (~10%) (192-194). Ribavirin is teratogenic, requiring strict adherence to birth control for both men and women receiving this drug.

The majority of adverse events occurring with PEG-IFN and ribavirin subside after cessation of treatment and can be managed with appropriate clinical monitoring and dose adjustments during therapy.

Page 46 of 309

Figure 1-2. Advances in hepatitis C therapy with respect to tolerability and efficacy

Abbreviations: BOC, boceprevir; DSV, dasabuvir; EBR, elbasvir; GZR, grazoprevir; IFN, interferon; LDV, ledipasvir; OMV, ombitasvir; PEG-IFN, pegylated interferon; PTV, paritaprevir; RBV, ribavirin; SIM, simeprevir; SOF, sofosbuvir; TVR, telaprevir; VEL, velpatasvir Reproduced with permission from (140), revised.

Pegylated interferon, ribavirin and 1st generation, 1st wave protease inhibitors While improving efficacy in comparison with PEG-IFN and ribavirin alone, the addition of a first-generation protease inhibitor to PEG-IFN and ribavirin combination therapy has generally resulted in significant additional toxicity (200-204) (Figure 1-2). In clinical trials of telaprevir in combination with PEG-IFN α-2a and ribavirin, marked additional dermatological (rash, pruritus), gastrointestinal (nausea, diarrhoea) and haematological (anaemia) toxicity in the triple therapy arms was demonstrated (205-207). While rare, fatal and non-fatal serious skin reactions, including Stevens-Johnson Syndrome, drug reaction with eosinophilia and systemic symptoms, toxic epidermal necrolysis and erythema multiforme, have been reported in individuals receiving telaprevir, both in clinical trials (<1%) and in post-marketing experience. The addition of boceprevir to PEG-IFN and ribavirin also resulted in marked additional haematological toxicity, largely anaemia (208, 209).

Page 47 of 309 Interferon-free direct-acting antiviral regimens Compared with PEG-IFN and ribavirin, DAAs have a remarkably clean safety and side-effect profile, resulting in dramatic improvements in tolerability (Figure 1.2). Fatigue, nausea, and headache were the only side-effects more common than placebo for sofosbuvir (210) and ombitasvir/paritaprevir/ritonavir with dasabuvir (211), with most adverse events in interferon- free regimens attributed to ribavirin (212, 213). In an analysis of HCV mono-infected adults who received PEG-IFN, ribavirin and boceprevir (n=97) or sofosbuvir and ribavirin (n=60), a significantly higher frequency of treatment discontinuations due to adverse events (35% vs 0%), anaemia (55% vs 7%), neutropenia (44% vs 0%) and thrombocytopenia (45% vs 0%) was demonstrated with PEG-IFN, ribavirin and boceprevir as compared with sofosbuvir and ribavirin (214).

Despite improvement in tolerability, specific toxicity concerns do warrant discussion.

Numerous and complex drug-drug interactions are possible with HCV DAAs. An awareness of pharmacokinetics and potential drug-drug interactions is required in order to prevent morbidity and ensure treatment efficacy. All approved DAAs interact with CYP450 enzymes or transporters, including P-glycoprotein (P-gp), organic anion-transporting polypeptide (OATP) and breast cancer resistance protein (BRCP). In general, the NS3/4a protease inhibitors and NS5A assembly inhibitors demonstrate the greatest potential for clinically significant drug-drug interactions, with metabolism by, and inhibition or induction of, one or more CYP450 enzymes, primarily CYP3A4, and bi-directional interactions with transporters, including BRCP, OATP and P-gp. In contrast, sofosbuvir appears to have little potential for clinically significant drug- drug interactions; while sofosbuvir is a substrate of both P-gp and BRCP, it does not induce or inhibit CYP450 enzymes. Clinically relevant pharmacokinetic properties of approved HCV DAAs, which may be implicated in the development of toxicity, including drug-drug interactions, are included in the Supplementary Appendix. The potential for drug-drug interactions should be considered in all people undergoing treatment with HCV DAAs, with a thorough risk assessment prior to and during DAA therapy.

Based on post-marketing surveillance, the US FDA have issued a warning regarding the risk of life-threatening bradyarrhythmias in individuals taking amiodarone in combination with either of two sofosbuvir-containing regimens, sofosbuvir/ledipasvir and sofosbuvir+ simeprevir. This followed reports of one patient's death following bradyarrhythmia, and three cases of patients developing complete heart block and requiring pacemaker insertion (215). Subsequently, three cases of bradyarrhythmia requiring pacemaker insertion in individuals with cirrhosis receiving sofosbuvir plus daclatasvir or simeprevir (with or without ribavirin) were reported in France

Page 48 of 309 (216). To date, nine cases of symptomatic bradycardia have been reported worldwide, with most receiving concomitant amiodarone. Bradycardia has generally occurred within hours to days, but cases have been observed up to 2 weeks after initiating HCV treatment. Bradycardia appears to resolve after discontinuation of therapy. The mechanism by which this occurs is uncertain. Amiodarone co-administration is not recommended with sofosbuvir-containing regimens. If co-administration is necessary, initiation of HCV antiviral therapy should occur under direct supervision with cardiac monitoring.

NS3/4A protease inhibitors are not recommended in individuals with severe (Child-Pugh Class C) hepatic impairment. There have been post marketing reports of hepatic decompensation, hepatic failure, and death in individuals with advanced or decompensated cirrhosis receiving DAA regimens containing simeprevir and paritaprevir (211, 217).

Drug-induced liver injury may be of concern with specific DAA regimens. On-treatment elevations in ALT have been noted in individuals receiving ombitasvir/paritaprevir/ritonavir and dasabuvir (with or without ribavirin) and asunaprevir + daclatasvir. In the case of ombitasvir/paritaprevir/ritonavir and dasabuvir, elevations of ALT greater than five times the upper limit of normal (ULN) occurred in approximately 1% of individuals enrolled in phase II and III clinical trials (211). These ALT elevations were typically asymptomatic, occurred during the first four weeks of treatment and declined within two to eight weeks of onset with continued administration of the HCV antiviral drugs. ALT elevations were significantly more frequent in women who were using ethinyl estradiol-containing . ALT elevations in association with asunaprevir + daclatasvir combinations occurred with variable frequency (2- 10%) in clinical trials and infrequently led to treatment discontinuation (218-221). ALT elevations appear to be more frequent in studies of Japanese populations (221) as compared with US or European cohorts (218, 219). Further, drug-induced liver injury with systemic and immuno-allergic features (fever, jaundice, eosinophilia) has been reported in individuals receiving asunaprevir + daclatasvir (222, 223). As such, more frequent monitoring is required with liver function tests recommended at least once every two weeks for the first 12 weeks of treatment, followed by every four weeks thereafter (224).

Ribavirin-free direct-acting antiviral regimens Ribavirin-free DAA regimens offer the cleanest safety profile, exemplified by analysis of pooled data from Phase II and III trials of grazoprevir/elbasvir and ledipasvir/sofosbuvir co- administered with and without ribavirin (Table 1-9) (225-227). In phase III studies of the once- daily fixed-dose combination tablet of ledipasvir/sofosbuvir with and without ribavirin (n=1952), treatment-related adverse events occurred in 71% and 45% of individuals treated with

Page 49 of 309 and without ribavirin, respectively, with the most commonly reported adverse events in both groups being fatigue, headache, nausea and insomnia (225). Most adverse events in both treatment groups were mild or moderate in severity. Serious adverse events were reported by 34 individuals (3%) who received ledipasvir/sofosbuvir with ribavirin and by 17 (2%) who received ledipasvir/sofosbuvir alone (225). In pooled analysis of phase II and III trials of grazoprevir/elbasvir, in those individuals receiving grazoprevir/elbasvir alone (n=1033), the only adverse events reported in more than 5% of the study population were fatigue and headache (227). In the pivotal phase III trials of the pan-genotypic regimen, sofosbuvir/velpatasvir (228), no difference in adverse event profile was noted between those who received sofosbuvir/velpatasvir and those who received placebo for 12 weeks. The most common adverse events in both groups were headache, fatigue, nasopharyngitis and nausea (Table 1-10) (228).

Page 50 of 309 Table 1-9. Adverse events reported with grazoprevir/elbasvir (with and without ribavirin), compared with placebo

GZR/EBR + GZR/EBR Placebo Safety parameter RBV N=1033 N=105 N=657 Clinical adverse event Any adverse event 71% 84% 69% Treatment-related adverse event 40% 68% 39% Serious adverse events Serious adverse event 2% 3% 3% Treatment-related serious adverse event 0.1% 0.5% 0% Adverse events (>5% study population) Fatigue 12% 25% 10% Headache 12% 16% 9% Nausea - 13% - Asthenia - 9% - Anemia - 9% - Insomnia - 9% - Pruritus - 9% 7% Rash 7% - Dyspnoea - 6% - Laboratory parameters Hb <100 g/L 0% 3% 0% Grade 3-4 elevation in ALT 2% 1% 9% Grade 3-4 elevation in bilirubin 0.3% 6% 0% Abbreviations: ALT, alanine aminotransferase; EBR, elbasvir; GZR, grazoprevir; Hb, haemoglobin; RBV, ribavirin

Table 1-10. Adverse events reported with sofosbuvir/velpatasvir compared with placebo

Placebo Sofosbuvir/Velpatasvir Safety Parameter N = 116 N = 624 Clinical adverse event Any adverse event 77% 78% Grade 3 or 4 adverse event <1% 3% Discontinuation for adverse event 2% <1% Serious adverse events Serious adverse events 0% 2% Death 0% <1%* Adverse events (≥10% study population) Headache 28% 29% Fatigue 20% 20% Nasopharyngitis 10% 13% Nausea 11% 12% Laboratory parameters Hb <100 g/L 0% <1% Grade 3 or 4 laboratory adverse event 12% 7% *Death: 1 individual died during sleep 8 days after treatment completion; deemed unrelated to study drug Abbreviations: Hb, haemoglobin

Page 51 of 309 Direct-acting antiviral efficacy in chronic HCV infection among PWID and people with HIV/HCV coinfection The rising public health burden of HCV infection has occurred in the setting of sub-optimal cure rates and treatment-related toxicity with interferon-containing therapy, which has resulted in both limited uptake and adverse individual outcomes. The advent of highly effective, well tolerated interferon-free DAA therapy has revolutionised HCV therapeutics (140), with daily fixed-dose combination DAA regimens providing cure in greater than 90% of individuals with chronic infection (Figure 1.2) (228-230). The availability of DAA therapy has given rise to significant therapeutic optimism, providing an opportunity for broad treatment scale-up with the potential for HCV elimination, particularly among marginalised or “high-risk” populations, including PWID and people with HIV/HCV co-infection (75, 231-233).

When assessing suitability for interferon-based HCV therapy, certain populations had been considered “high-risk”, given concerns regarding efficacy, toxicity and adherence. While HCV treatment in PWID is feasible and successful across a broad range of multidisciplinary healthcare settings (131, 132, 137, 234), interferon-based treatment uptake was low with multiple barriers to care at an individual and systems level, driven by concerns regarding adherence, social instability, treatment-related adverse effects and psychiatric comorbidity (235). In people with HIV/HCV co-infection, IFN-based therapy has had limited success, with concerns regarding efficacy and tolerability. Therapy with pegylated-interferon and ribavirin resulted in SVR in less than 30% of HIV-positive individuals with HCV genotype 1 infection (134, 135). With the addition of first-generation HCV NS3/4a protease inhibitors, efficacy improved, but additional adverse events and drug-drug interactions further complicated therapy (203, 236).

Evidence from clinical trials Phase II and III interferon-free DAA clinical trials have been undertaken in various populations, previously deemed “high-risk”, including people receiving OST (including recent PWID) and people with HIV/HCV co-infection. Overall efficacy and safety data have been similar to that seen in general populations.

HIV/HCV co-infection In contrast to results achieved with interferon-based therapy, HIV co-infection does not appear to compromise DAA efficacy (Table 1-11). Phase III trials have demonstrated high sustained virological response at 12 weeks post-treatment (SVR12), equivalent to that observed in HCV

Page 52 of 309 mono-infection (229, 237-240). Following 12 weeks of interferon-free, ribavirin-free DAA therapy, SVR12 was achieved in 95%-97% of HIV/HCV co-infected participants receiving sofosbuvir/ledipasvir (229), sofosbuvir plus daclatasvir (237), grazoprevir/elbasvir (239) and sofosbuvir/velpatasvir (240).

However, the generalisability of these clinical trial results has been questioned, in large part due to the exclusion criteria pertaining to antiretroviral therapy, active drug use, CD4 T-cell count and detectable HIV RNA. Saeed et al (241) reviewed Phase III DAA clinical trial eligibility and compared this with the demographic, behavioural and clinical characteristics of patients within the Canadian Coinfection Cohort (n=874). They demonstrated that a minority (6%– 10%) of HIV/HCV co-infected patients would have been eligible for enrolment in the majority of HIV/HCV Phase III trials, largely related to prescription of specific antiretroviral agents (drug–drug interactions) and active drug use. ALLY-2 (237), which assessed the efficacy and safety of sofosbuvir and daclatasvir, was the exception, with 43% of HIV/HCV co-infected patients potentially eligible for inclusion. Drug and alcohol use per se was not an exclusion criteria, with “active substance abuse” only regarded as exclusionary if the investigator deemed it “inappropriate.” The study by Saeed et al (241) is important in highlighting the potential gap between clinical trial–based efficacy and “real-world” impact.

Page 53 of 309 Table 1-11. Phase II and III trials of interferon-free DAA regimens in people with HIV/HCV co-infection

Trial phase Study population and key HCV Duration Author, year DAA regimen N SVR12 Trial acronym inclusion/exclusion criteria GT (weeks) 12 GT2: 88% (242) Sulkowski, Phase III CD4>200, HIV RNA<50 1 2 SOF + RBV 1-3 223 GT1: 76% 2014 PHOTON-1 Exclusion: Active drug use 24 GT3: 94% 12 GT2: 88% (243) Molina, Phase III CD4>200, HIV RNA<50 1 2 SOF + RBV 1-4 274 GT1: 85% 2015 PHOTON-2 Exclusion: Active drug use 24 GT3: 89% GT4: 84% (244) Osinusi, Phase II CD4>100, HIV RNA<50 1 SOF/LDV 1 50 12 98% 2015 ERADICATE (229) Naggie, Phase III CD4>100, HIV RNA<50 1 SOF/LDV 1 335 12 96% 2015 ION-4 Exclusion: Active drug use 2 (237) Wyles, Phase III 1 8 76% CD4>200, HIV RNA<50 SOF + DCV 1-4 203 2015 ALLY-2 12 97% (238) Sulkowski, Phase III CD4>200, HIV RNA<40 1 12 94% PrO + D +/- RBV 1 63 2015 TURQUOISE-I Exclusion: Active drug use 2 24 91% (245) Sulkowski, Phase II CD4>300, HIV RNA<20 1 GZR/EBR 87% 1 59 12 2015 C-WORTHY Exclusion: Active drug use GZR/EBR + RBV 97% (239) Rockstroh, Phase III CD4>200, HIV RNA<20 1 GZR/EBR 1, 4, 6 218 12 96% 2015 C-EDGE COINFECTION Exclusion: Drug use within 12 months (240) Wyles, Phase III CD4>100, HIV RNA<50 1 SOF/VEL 1-4 106 12 95% 2016 ASTRAL 5 Exclusion: Active drug use 2 1 CD4 T-cell count >200 cells/mm3 (>100 cells/mm3 in ION-4, >300 cells/mm3 in C-WORTHY) and HIV RNA <50 copies/mL (<40 copies/mL in TURQUOISE-1) if on combination antiretroviral therapy (>90% of study population) or CD4 T-cell count >500 cells/mm3 if not on combination antiretroviral therapy 2 Active drug or alcohol use at enrolment: History of clinically relevant drug or alcohol use within 6-12 months of screening, confirmed by urine drug screen at screening

Abbreviations: DCV, daclatasvir; D, dasabuvir; GT, genotype; GZR/EBR, grazoprevir/elbasvir; PrO, paritaprevir/ritonavir/ombitasvir fixed dose combination; RBV, ribavirin; SOF, sofosbuvir; SOF/LDV, sofosbuvir/ledipasvir fixed dose combination; SOF/VEL, sofosbuvir/velpatasvir; SVR, sustained virological response

Page 54 of 309 People who inject drugs Evidence from clinical trials with interferon-containing regimens indicated that recent drug use did not compromise HCV treatment outcomes (246). Phase II and III trials of DAA therapy have demonstrated similar high efficacy among people receiving OST as compared with people not receiving OST (Table 1-12) (247-250). However, in large part, these individuals have been stable on OST and people reporting recent drug use have been excluded, either by time since last reported drug use or by urine drug screen.

A post hoc analysis of treatment efficacy and safety was performed among people who use drugs (PWUD) while receiving treatment in the ION-1 studies, in which participants with chronic HCV genotype 1 received sofosbuvir/ledipasvir (with or without ribavirin) for 8-24 weeks (249). As a positive urine drug test at screening was exclusionary, in order to assess drug use on treatment, stored samples were retrieved and tested retrospectively. Drug use during therapy was seen in 23% (n=70), with the majority of positive samples detecting cannabinoids (19%). Use of opiates (1%), cocaine (1%) or amphetamine/methamphetamine (<1%) was demonstrated in a minority. High treatment efficacy and adherence was seen, regardless of on- treatment drug use (any drug use on-treatment: SVR12 97%, 68/70; no drug use on-treatment: SVR12 99%, 652/657).

In the C-EDGE COSTAR trial, the efficacy and safety of grazoprevir/elbasvir for 12 weeks in chronic HCV genotypes 1, 4 and 6 was assessed among people receiving OST (n=301), the majority of whom had ongoing drug use during treatment and follow-up (251). Active drug use was assessed by regular urine drug screen; 58% had a positive urine drug screen at treatment initiation with stable patterns of drug use throughout treatment. At treatment initiation in the immediate treatment group, active use of benzodiazepines, opiates, cocaine or amphetamines/methamphetamines was seen in 25%, 22%, 10% and 5%, respectively. Overall SVR12 was 92%, with similar treatment adherence and efficacy to the other C-EDGE phase III studies that excluded people with recent drug use (239, 252).

To date, no studies have reported outcomes among recent PWID who are not receiving OST.

Page 55 of 309 Table 1-12. Phase II and III trials of interferon-free DAA regimens in people who inject drugs and people receiving opioid substitution therapy

Study population and key Trial phase HCV Duration Author, year inclusion/exclusion DAA regimen N SVR12 Trial acronym GT (weeks) criteria Phase II: AVIATOR, M14-103 OST (247) Puoti, GT1a: 96% Phase III: PEARL II–IV, Exclusion: Active drug use PrO + D +/- RBV 56 1 12 – 24 2014 GT1b: 100% SAPPHIRE I-II, TURQUOISE-II 1 OST (248) Lalezari, Phase II Exclusion: Active drug use PrO + D +/- RBV 38 1 12 97% 2015 - 1 PWUD 2 (249) Grebely, Phase III OST 196 PWUD: 97% SOF/LDV +/- RBV 1 8 – 24 2016 ION-1 Exclusion: Active drug use 70 OST: 94% 1 OST (250) Grebely, Phase III Exclusion: Active drug use SOF/VEL 51 1-4 12 96% 2016 ASTRAL 1-3 1

(251) Dore, Phase III OST GZR/EBR 301 1, 4, 6 12 92% 2016 C-EDGE COSTAR (Current PWUD 58%)

1 Active drug or alcohol use at enrolment: History of clinically relevant drug or alcohol use within 6-12 months of screening, confirmed by urine drug screen at screening 2 PWUD on treatment: Stored serum samples from participants enrolled in ION-1 collected on-treatment (weeks 8 and 12) tested retrospectively testing for the following illicit drugs: amphetamines, methamphetamines, barbiturates, benzodiazepines, cocaine, methadone, opiates, oxycodone, phencyclidine, propoxyphene and cannabinoids.

Abbreviations: D, dasabuvir; GT, genotype; GZR/EBR, grazoprevir/elbasvir; OST, opioid substitution therapy; PrO, paritaprevir/ritonavir/ombitasvir fixed dose combination; RBV, ribavirin; SOF, sofosbuvir; SOF/LDV, sofosbuvir/ledipasvir fixed dose combination; SOF/VEL, sofosbuvir/velpatasvir; SVR, sustained virological response

Page 56 of 309 Evidence from “real world” cohorts Observational cohorts that have evaluated DAA regimens among broader populations in the “real world” have generally shown favourable treatment outcomes (253-260). In the large US Veterans Affairs cohort (253), a high proportion of people with chronic HCV genotypes 1-4 achieved SVR (91%) following, predominately sofosbuvir-containing, DAA therapy initiated between January 2014 and June 2015. The cohort included individuals with a history of substance use disorder (37%), history of alcohol use disorder (44%) and HIV co-infection (4%). A history of “alcohol use disorder” did not impact SVR. HIV co-infection and history of substance use disorder were not included in the model.

The “real world” efficacy of DAA therapy among people with HIV/HCV co-infection appears to be comparable to clinical trial efficacy (256-259, 261). In a population traditionally designated difficult to treat, given poor outcomes with pegylated interferon and ribavirin (SVR ≤30%) (262, 263), high SVR12 (93%) was demonstrated among people with HIV/HCV co- infection and cirrhosis enrolled in the French National Agency for Research on AIDS and Viral Hepatitis CO13 HEPAVIH cohort (258). Of 189 treated (75% male, 58% HCV genotype 1, 16% Child-Pugh class B or C ), the vast majority received 24 weeks of sofosbuvir in combination with an NS5A inhibitor, daclatasvir (65%) or ledipasvir (12%), with or without ribavirin.

Similarly high SVR has been reported in cohorts of PWUD. In a single-centre US cohort of PWUD (defined as people receiving OST, people reporting recent drug use, or people with a positive urine drug screen) treated with interferon-free DAA therapy, SVR12 was 96% (44/46), compared with 95% (41/43) in non-PWUD (264). Most of the cohort reported recent drug use (65%) and receipt of OST (78%). In a small single-centre Austrian cohort of active PWID (n=15), directly-observed DAA therapy was initiated in concert with OST (265). SVR12 were achieved in 100%, with excellent adherence (100%). While encouraging, further research is required to determine the feasibility and efficacy of DAA therapy in broader populations of recent PWID.

Page 57 of 309 Reinfection following HCV treatment One challenge in achieving HCV elimination is reinfection. There is concern that HCV reinfection may compromise the individual and population level benefits of HCV treatment in populations with ongoing risk behaviour (266). The risk of reinfection is cited as a reason for not offering treatment to PWID (266, 267). However, the incidence of reinfection following interferon-based treatment for chronic HCV is low among lifetime PWID (246, 268). Conversely, high reinfection incidence has been reported in some cohorts of HIV-positive MSM (269, 270). Mathematical modelling suggests that substantial reductions in HCV incidence and prevalence could be achieved with targeted DAA therapy among those at highest risk of ongoing transmission (75, 271).

Who is at “high-risk” of reinfection? Defining the population of interest When assessing suitability for interferon-based HCV therapy, certain populations, including PWID, people who are incarcerated, people with HIV/HCV co-infection and MSM, had been considered at “high-risk” of reinfection (268). However, these populations are heterogeneous with different levels of risk attributable to specific subgroups.

Sub-populations of PWID include those who report injecting an illicit drug at least once (lifetime PWID), those who have ceased injecting drug use (former PWID) and those who continue to inject drugs (recent PWID, with definitions of “recent” varying between one to 12 months) (272). Among lifetime PWID, there also exists a group of people receiving opioid substitution therapy (OST), some of whom may be recent PWID. Understanding the definitions for different PWID populations is crucial to accurately define outcomes and reinfection risk following DAA therapy.

Similarly, not all people with HIV/HCV co-infection demonstrate contemporary behaviours placing them at “high risk”. The higher HCV reinfection incidence reported in some studies among people with HIV/HCV co-infection, as compared to HCV mono-infection, has been driven by cohorts of acute HCV infection in HIV-positive MSM (268). Caution should be taken not to extrapolate these reinfection rates to general HIV/HCV co-infected populations. While similar risk behaviours are observed in HIV-positive and HIV-negative MSM (273), HCV incidence is significantly higher among HIV-positive MSM (85). As DAA treatment scale-up occurs, a better understanding of drug use and sexual behaviours which pose a risk of reinfection are required.

Page 58 of 309 In a recent meta-analysis, Simmons et al (268) examined the risk of HCV recurrence following interferon-based treatment-induced SVR in three different populations, defined by their perceived risk of reinfection – HCV mono-infected “low risk” (no recognised risk factors for reinfection), HCV mono-infected “high risk” (recognised risk factors for reinfection: former or recent injecting drug use, incarceration, MSM) and HIV/HCV co-infection. Reinfection incidence was 0.0 per 100 py (95% 0.0, 0.0) in those deemed “low risk”, 1.9 per 100 py (95% CI 1.1, 2.8) in those deemed “high risk” and 3.2 per 100 py (95% CI 0.0, 12.3) in those with HIV/HCV co-infection. Regardless of risk category, reinfection incidence was low. However, it was unclear what proportion of those included in the “high risk” group continued to demonstrate behaviours following SVR which posed a risk for HCV reinfection. Despite being included in the definition, no studies of MSM were included in the “high risk” analysis. Also noteworthy, the higher reinfection incidence in the group with HIV/HCV co-infection was primarily driven by a single study of acute HCV infection in HIV-positive MSM (270).

A summary of primary studies and meta-analyses assessing HCV reinfection incidence following treatment in PWID and people with HIV/HCV co-infection is presented in Table 1-13, with the primary studies divided by duration of HCV infection. In general, HCV reinfection incidence in cohorts of acute or recent HCV infection is higher than in cohorts of chronic HCV infection, presumably given contemporary risk behaviours for HCV transmission in acute HCV cohorts. However, it should be noted that there is uncertainty around these reinfection estimates in primary studies due to small sample size, limited follow up, varied testing intervals, retrospective study designs, exclusion of recent PWID from trials, varied definitions for recent injection drug use and time at-risk for reinfection, and the inability to accurately distinguish relapse from reinfection. As a result, for the most part, few cases of reinfection have been observed, and variability in reinfection incidence estimates is high.

Page 59 of 309 Table 1-13. HCV reinfection incidence following treatment-induced clearance (2011–2016)

Subjects Reinfection Reinfection incidence/100 Author, year Population Location, study design (n) (n) py (95% CI) Meta-analysis studies: Reinfection post treatment for acute and chronic HCV infection (268) Simmons, 2016 “Low risk” 1 Meta-analysis (31 studies) 7969 4 0.0 (0.0, 0.0) (268) Simmons, 2016 “High risk” 1 Meta-analysis (14 studies) 771 36 1.9 (1.1, 2.8) (268) Simmons, 2016 HIV/HCV 1 Meta-analysis (4 studies) 309 31 3.2 (0.0, 12.3) (12) Hagan, 2015 HIV-positive MSM Meta-analysis (2 studies) 170 38 11.4 (7.4, 17.7) (246) Aspinall, 2013 PWUD Meta-analysis (5 studies) 131 7 2.4 (0.9, 6.1) Primary studies: Reinfection post treatment for chronic HCV infection (251) Dore, 2016 OST International; Prospective 301 6 4.6 (1.7, 10.0) (274) Midgard, 2016 PWID Norway; Prospective 94 12 2.0 (1.0, 3.5) (275) Weir, 2016 PWID Scotland; Retrospective 277 7 1.7 (0.7, 3.5) (276) Pineda, 2015 HIV, PWID Spain; Prospective 84 4 1.2 (0.3, 3.1) (277) Conway, 2013* Recent PWID Canada; Prospective 70 4 2.9 (1.1, 7.2) (278) Deshaies, 2013* Recent PWID Canada; Prospective 20 2 6.3 (1.7, 20.3) (279) Edlin, 2013* Recent PWID US; Not reported 15 1 2.2 (3.9, 11.5) (280) Hilsden, 2013* Recent PWUD Canada; Prospective 23 1 2.8 (0.0, 14.5) (281) Marco, 2013* Incarcerated Spain; Retrospective 119 9 5.3 (282) Ruzic, 2013* Former PWID Serbia; Retrospective – prospective 20 0 0 (0.0, 3.7) (283) Grady, 2012* Recent PWUD Netherlands; Prospective 42 1 0.8 (0.0, 3.7) (284) Manolakopoulos, 2012* PWID Greece; Retrospective 61 5 4.1 (1.8, 9.2) Primary studies: Reinfection post treatment for recent HCV infection (269) Lambers, 2011** HIV-positive MSM Netherlands; Retrospective 56 11 15.2 (8.0, 26.5) (285) Grebely, 2012 PWID, HIV-positive MSM Australia; Prospective 67 5 12.3 (5.1, 29.6) (270) Martin, 2013* ** HIV-positive MSM England; Retrospective 1142 27 9.6 (6.6, 14.1) *Studies included in meta-analysis performed by Simmons et al (268); ** Studies included in meta-analysis performed by Hagan et al (12) 1 Simmons et al (268) examined the risk of HCV recurrence following SVR in three different populations, defined by their risk of reinfection – HCV mono-infected “low risk” (no recognised risk factors for reinfection), HCV mono-infected “high risk” (recognised risk factors for reinfection: former or recent injecting drug use [12 studies] incarceration [2 studies], MSM [0 studies]) and HIV/HCV co-infection. 2 Documented primary HCV infection only Abbreviations: MSM, men-who-have-sex-with-men; OST, opioid substitution therapy; PWID, people who inject drugs; PWUD, people who use drug.

Page 60 of 309 HCV reinfection among people who inject drugs The risk of reinfection is cited as a reason for not offering treatment to PWID (267). However, the incidence of reinfection following interferon-based treatment for chronic HCV is generally low among people who have ever injected drugs (lifetime PWID, reinfection incidence: 0 – 5 per 100 py) (Table 1-13) (246, 268).

The risk of HCV reinfection is significantly higher in people treated for chronic HCV infection who report ongoing risk behaviour, with reinfection incidence in those reporting injecting drug use post treatment ranging between 0.0 – 33.0 per 100 py (246, 274, 275, 281, 285-288). In a meta-analysis examining reinfection incidence among PWUD (recent and lifetime), overall reinfection incidence was 2.4 per 100 py (95%CI 0.9, 6.1), rising to 6.4 per 100 py (95% CI 2.5, 16.7) in those who reported injecting drug use post SVR (246).

Several recent studies have demonstrated the impact of ongoing injecting drug use following treatment on reinfection incidence. Midgard et al (274) followed “former” PWID for up to seven years after successful treatment with pegylated-interferon and ribavirin in Norway (n=94). One of the inclusion criteria for initial study entry and interferon-based treatment was abstinence from injecting for more than six months; 39% reported injecting drug use during follow-up. In this cohort, 12 cases of reinfection were identified for an overall reinfection incidence of 2.0 per 100 py (95% CI 1.0, 3.5). Reinfection incidence rose to 5.8 per 100 py (95% CI 3.0, 10.2) among participants reporting injecting drug use post treatment. Similarly, Weir et al (275) examined reinfection incidence among lifetime PWID following SVR in Scotland (n=277), with seven cases of reinfection identified and an overall reinfection incidence of 1.7 per 100 py (95% CI 0.7, 3.5). Among PWID who had been hospitalised for an opiate or injection-related cause post SVR (11%, n=29), the risk of HCV reinfection was significantly higher (adjusted hazard ratio 12.9, 95% CI 2.2, 76.0, p=0.002) with an increase in reinfection incidence to 5.7 per 100 py (95% CI 1.8, 13.3).

Data is beginning to emerge on reinfection following treatment with DAA therapy. In the C- EDGE COSTAR trial among people receiving OST, six cases of reinfection were identified at or prior to post treatment week 24, with five cases of reinfection detected at post treatment week eight. Reinfection incidence was 4.6 per 100 py (95% CI 1.7, 10.0) (251). Urine drug screen was positive both during and following treatment in five of the six cases. Of note, reinfection cleared spontaneously in three of the six cases. No cases of reinfection were identified up to 24 weeks post treatment in the ION-1 or ASTRAL 1-3 trials (249, 250).

Page 61 of 309 HCV reinfection among HIV-positive MSM Reinfection rates in HIV-positive MSM following primary HCV infection are varied, with high incidence reported in acute HCV cohorts (269, 270) (Table 1-13). A meta-analysis examining reinfection HCV incidence among HIV-positive MSM who denied injecting drug use reported a reinfection incidence of 11.4 per 100 py (95% CI 7.4, 17.7) (12), though results were pooled from only two retrospective acute HCV cohorts in Europe (269, 270). Ingiliz et al (289) analysed reinfection incidence in a large cohort of HIV-positive MSM in Western Europe (n=552), with 143 reinfections demonstrated over 1952 py of follow-up. Overall reinfection incidence was 7.3 per 100 py (95% CI 6.2, 8.6) with 25% reinfected at 3 years. Multiple reinfections were noted in some individuals (second reinfection, n=69; third reinfection, n=13) for a secondary reinfection incidence of 18.8 per 100 py (95% CI 12.9, 27.5). Importantly, the results combined two different populations at risk for reinfection, those with treatment-induced clearance and those with spontaneous clearance. Individuals with acute HCV infection and spontaneous clearance appear to be at higher risk of reinfection (53, 56, 104) than those with chronic HCV infection and treatment-induced SVR, indicative of different rates of ongoing risk behaviour (268).

Reinfection has been documented in Phase II and III DAA trials among people with HIV/HCV co-infection, in some cases prior to the primary endpoint of the study (SVR12). In TURQUOISE-I (238), two cases of reinfection were detected after the participants achieved SVR12; high risk sexual behaviour was reported. In C-EDGE COINFECTION (239), two cases of reinfection were documented prior to SVR12; risk factors for reinfection were not reported.

Populations at high risk of reinfection, such as PWID and HIV-positive MSM, are not mutually exclusive. While often discussed as separate cohorts, it is important to remember that there is significant overlap (290). Among a cohort of HIV/HCV co-infected individuals (of whom 86% reported ever injecting drugs), overall reinfection incidence was 1.2 per 100 py (95% CI 0.3, 3.1) (276). Reinfection incidence in those individuals who used heroin and/or cocaine during follow-up was 8.7 per 100 py (95% CI 4.8, 23.7). High reinfection incidence following treatment for HCV infection in individuals with ongoing high risk behaviour emphasises the need for post-treatment surveillance, harm reduction strategies and education.

Page 62 of 309 Mathematical modelling: Treatment-as-Prevention and the impact of reinfection The burden of disease attributed to HCV is high among PWID, and is increasing among HIV- infected MSM. The potential for broad access to interferon-free DAA regimens has stimulated discussion around HCV Treatment-as-Prevention. Mathematical modelling suggests that substantial reductions in HCV incidence and prevalence could be achieved by targeted DAA treatment scale-up amongst those at highest risk of ongoing transmission (75, 271, 291, 292). Using HCV treatment uptake data from seven sites in the United Kingdom, Martin et al demonstrated that treating 26 per 1000 HCV-infected PWID per annum with DAA therapy could achieve a 15-50% decrease in chronic HCV prevalence within 10 years (292). Despite the high cost of DAA therapy, treating recent PWID and HIV-positive MSM with early liver disease appears to be cost-effective compared to delaying until cirrhosis, given the reduction in liver-related complications and additional benefit of averting secondary infections (232, 233, 293).

HCV treatment scale-up could have a population level prevention benefit by reducing the size of the HCV viraemic reservoir. However, ongoing risk behaviour following successful HCV therapy will contribute to reinfection. As shown in Figure 1-3, rapid scale-up of DAA therapy (>8% per year) among PWID markedly increases the aviraemic proportion and as such, increases the proportion susceptible to HCV reinfection (294). While initially this leads to an increase in the number of people with HCV reinfection, as the viraemic prevalence decreases overall, the number of people with reinfection also decreases. A slower scale-up of DAA therapy (≤4% per year) has more limited impact on the viraemic prevalence and reinfection.

250 3,500

3,000 Treated Number Annual 200 2,500

150 2,000

100 1,500 1,000

Secondary Infections 50 500

0 0

Treat 1% Treat 2% Treat 4% Treat 8% Treat 10% Treat 1% Treat 2% Treat 4% Treat 8% Treat 10%

Figure 1-3. Rate of HCV reinfection following HCV treatment scale-up assuming various rates of treatment uptake

Each line represents the expected number of individuals with HCV reinfection (left axis) in subsequent years based on an assumed annual treatment rate scenario. Permission to reproduce the image has been obtained from the author (294).

Page 63 of 309

Prioritizing treatment to those at risk of HCV transmission, including recent PWID and HIV- positive MSM engaging in high risk behaviour, is consistent with international guidelines (91, 103, 295, 296). Using data from the United Kingdom Collaborative HIV Cohort (UK CHIC), mathematical modelling predicted that over the next decade 94% of HCV infections in HIV- positive MSM would occur among high-risk individuals. As such, the greatest impact on HCV incidence and prevalence would be achieved if DAA treatment scale-up was prioritised to those with recently diagnosed (<1 year) HCV infection and occurred in combination with behavioural interventions (75). Similarly, modelling within the Swiss HIV Cohort Study (SHCS) suggested that DAA treatment scale-up will need to occur in concert with behavioural risk reduction or stabilisation to achieve reductions in HCV incidence and prevalence (297). Stabilisation of rates of high-risk behaviour combined with increased treatment uptake and the use of DAAs was predicted to reduce HCV incidence by 77% (from 2.2 per 100 py in 2015 to 0.5 per 100 py in 2030) and prevalence by 81% (from 4.8% in 2015 to 0.9% in 2030). However, most importantly, the model highlighted that a decrease in high-risk behaviour alone could rapidly reduce HCV incidence, independent of treatment uptake or efficacy. Modelling estimates support broad access to DAA therapy, without limitations based on duration of infection, disease stage or drug use, to gain the greatest individual and population level benefits.

Page 64 of 309 Thesis rationale and objectives The management of recent (acute or early chronic) HCV infection is not standardised, with uncertainty regarding the optimal regimen and treatment duration, particularly as the HCV therapeutic landscape evolves with the advent of DAA therapy (298). Much of what is known about the timing of treatment initiation, regimen choice and duration of therapy in acute HCV infection comes from small observational studies and randomized controlled trials in selected populations with limited data on treatment in PWID and HIV co-infection. Individuals with diagnosed recent HCV infection are keen to consider treatment (133), and as such, this initial assessment may represent an ideal opportunity for therapeutic intervention. As interferon-free DAA therapy is established as the standard-of-care for chronic HCV infection (91, 103, 140), its role and activity in recent HCV infection requires evaluation. Shorter treatment durations should aim to optimise adherence and enhance cost-effectiveness. With interferon-based regimens, 12 weeks treatment is sufficient to cure greater than 90% of individuals with symptomatic acute HCV infection (298), as compared with 24-48 weeks treatment for chronic HCV infection.

Given the global burden of HCV-related disease among PWID and people with HIV, strategies to enhance HCV assessment, treatment and prevention are urgently needed. While detection is difficult, recognition of those with recent HCV infection may have individual and population level benefits. Diagnosis of acute HCV infection permits estimation of annual incidence rates and transmission patterns, facilitating implementation and review of prevention programs. Early diagnosis speeds linkage to multidisciplinary care, including education and counselling which may reduce high-risk behaviours (299) and subsequent transmission. Successful treatment of acute HCV infection in PWID and HIV-positive MSM, when combined with implementation of harm reduction strategies, should prevent transmission (Treatment-as- Prevention) and contribute to HCV elimination at a population level (75, 300).

Page 65 of 309 This thesis consists of a literature review (in this chapter) and four chapters (chapter two to five) describing original research findings. The specific aims of the research described in this thesis and relevant hypotheses are:

1. To evaluate the efficacy and safety of response-guided interferon-based therapy in individuals with recent HCV infection, by the proportion of treated participants (intention-to-treat [ITT] population) demonstrating a sustained virological response at 12 weeks post treatment (SVR12). Hypothesis: Response-guided therapy will be effective and safe in most individuals with recent HCV infection.

2. To evaluate the feasibility, efficacy and safety of ultra-short duration interferon-free therapy in individuals with recent HCV infection, by the proportion of treated participants (ITT population) demonstrating SVR12. Hypothesis: Short duration interferon-free therapy with sofosbuvir and ribavirin will be safe and effective in most individuals with recent HCV infection.

3. To evaluate the efficacy of response-guided interferon-containing and short duration interferon-free therapy in HCV mono-infection as compared with HIV/HCV co- infection Hypothesis: Short duration and/or response-guided therapy will be effective in most individuals with recent HCV infection, regardless of HIV serostatus.

4. To evaluate treatment adherence in individuals receiving response-guided interferon- containing and short duration interferon-free therapy with recent HCV infection Hypothesis: Short duration therapy will enhance treatment adherence.

5. To calculate the incidence of HCV reinfection among individuals treated for recent HCV infection. Hypothesis: HCV reinfection following treatment-induced clearance will occur in the context of ongoing risk behaviour

6. To assess the clinical significance of drug-drug interactions between HCV DAAs and HIV antiretroviral therapy. Hypothesis: Drug-drug interactions will occur and will impact on DAA prescribing in HIV/HCV co-infection.

Page 66 of 309 Chapter 2 The efficacy, safety and feasibility of response-guided interferon-based therapy in recent HCV infection

Chapter Introduction and Objectives The availability of HCV direct-acting antiviral therapy signalled a major advance in clinical medicine. The first generation HCV protease inhibitors, telaprevir and boceprevir, were approved by the US Food and Drug Administration for use in combination with pegylated- interferon and ribavirin in 2011 for treatment of chronic HCV infection.

Enhanced responsiveness with interferon-based therapy in recent HCV infection has allowed treatment duration to be shortened in comparison with chronic HCV infection; 12 weeks of interferon-based therapy appeared sufficient to cure greater than 90% of individuals with symptomatic acute HCV infection, as compared with 24-48 weeks treatment for chronic HCV infection. Shorter treatment durations should aim to optimise adherence, reduce toxicity and enhance cost-effectiveness. As with chronic HCV infection, response-guided interferon-based therapy may be effective in recent HCV infection.

The Australian Trial in Acute Hepatitis C II (ATAHC II; 2011-2015) evaluated the efficacy and safety of response-guided therapy with pegylated-interferon alfa-2a and weight-based ribavirin for individuals with recent HCV infection (estimated duration of infection <18 months). The Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C (DARE-C I; 2013- 2015), a sub study of ATAHC II, assessed the efficacy and safety of response-guided therapy with PEG-IFN alfa-2a, weight-based ribavirin and telaprevir for individuals with recent genotype 1 HCV infection (estimated duration of infection 6-18 months). A total of 82 participants (62% HIV-positive) were enrolled in ATAHC II (treated, n=52) and 14 (79% HIV- positive) in DARE-C I. SVR12 was 71% in both ATAHC II (37/52) and DARE-C I (10/14). Significant haematological toxicity was demonstrated with the addition of telaprevir.

While in ATAHC II and DARE-C I, the majority of participants with recent HCV infection were able to receive short duration (8-16 weeks) therapy, toxicity would limit implementation; the interferon-containing regimens used have been superseded by more tolerable interferon-free direct-acting antiviral regimens. The applicability of an interferon-containing strategy in the management of HCV infection is limited to settings in which direct-acting antiviral therapy is unavailable or very heavily restricted.

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The manuscript has been published in Antiviral Therapy.

Publication Martinello M, Hellard M, Shaw D, Petoumenos K, Applegate T, Grebely J, Yeung B, Maire L, Iser D, Lloyd A, Thompson A, Sasadeusz J, Haber P, Dore GJ, Matthews GV. Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II and DARE-C I studies. Antivir Ther. 2016;21(5):425-34. doi: 10.3851/IMP3035.

Addendum Martinello M, Hellard M, Shaw D, Petoumenos K, Applegate T, Grebely J, Yeung B, Maire L, Iser D, Lloyd A, Thompson A, Sasadeusz J, Haber P, Dore GJ, Matthews GV. Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II and DARE-C I studies. Antivir Ther. 2016;21(5):465. doi: 10.3851/IMP3073.

Page 68 of 309 Declaration I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Marianne Martinello

Viral Hepatitis Clinical Research Program Kirby Institute, UNSW Australia Wallace Wurth Building, Sydney NSW 2052 t: +61 413 276 968 e: [email protected]

Page 69 of 309 Co-authorship Acknowledgement In the case of Chapter Two, the nature and extent of my contribution to the work was the following:

Author name Contribution (%) Nature of contribution Conducted the data analysis, contributed to data Marianne Martinello 50 collection and led the development, writing and critical revision of the manuscript Contributed to study design, implementation, Margaret Hellard 2 data collection and critical revision of the manuscript Contributed to study design, implementation, David Shaw 2 data collection and critical revision of the manuscript Contributed to study design and statistical Kathy Petoumenos 2 analysis of data Contributed to study design and critical revision Tanya Applegate 2 of the manuscript Contributed to study design and critical revision Jason Grebely 2 of the manuscript Coordinated implementation of the study and Barbara Yeung 1 data collection. Contributed to revision of the manuscript. Coordinated implementation of the study and Laurence Maire 1 data collection. Contributed to revision of the manuscript. Contributed to study design, implementation, David Iser 2 data collection and critical revision of the manuscript Contributed to study design and critical revision Andrew Lloyd 1 of the manuscript Contributed to study design, implementation, Alex Thompson 2 data collection and critical revision of the manuscript Contributed to study design, implementation, Joe Sasadeusz 2 data collection and critical revision of the manuscript Contributed to study design and critical revision Paul Haber 1 of the manuscript Contributed to study design, data collection, Gregory J Dore 15 interpretation of findings and critical revision of the manuscript Contributed to study design, data collection, Gail V Matthews 15 interpretation of findings and critical revision of the manuscript

Page 70 of 309 Short duration response-guided treatment is effective for most individuals with recent hepatitis C infection: the ATAHC II and DARE-C I studies

Marianne Martinello1, Margaret Hellard2,3,4, David Shaw5, Kathy Petoumenos1, Tanya Applegate1, Jason Grebely1, Barbara Yeung1, Laurence Maire1, David Iser6, Andrew Lloyd7, Alexander Thompson6, Joe Sasadeusz8, Paul Haber9,10, Gregory J Dore1,11, Gail V Matthews1,11

1. The Kirby Institute, UNSW Australia, Sydney, NSW, Australia 2. Burnet Institute, Melbourne, VIC, Australia 3. Infectious Disease Unit, Alfred Hospital, Melbourne, VIC, Australia 4. Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, VIC, Australia 5. Royal Adelaide Hospital, Adelaide, SA, Australia 6. St Vincent’s Hospital, Melbourne, VIC, Australia 7. UNSW Australia, Sydney, NSW, Australia 8. Royal Melbourne Hospital, Melbourne, VIC, Australia 9. Royal Prince Alfred Hospital, Camperdown, NSW, Australia 10. University of Sydney, Sydney, NSW, Australia 11. St Vincent’s Hospital, Sydney, NSW, Australia

Page 71 of 309 Abstract Background and Objectives: Individuals with recent hepatitis C virus infection may benefit from shortened duration therapy. These studies evaluated the efficacy and safety of response- guided regimens with pegylated-interferon alfa-2a and ribavirin for people with recent HCV infection.

Methods: Participants with recent hepatitis C (duration of infection ≤18months) enrolled in the ATAHC II (pegylated-interferon alfa-2a +/- ribavirin) and DARE-C I (pegylated-interferon alfa-2a, ribavirin and telaprevir) studies were included for analysis. Treatment duration was response-guided (ATAHC II: 8, 16, 24 or 48 weeks; DARE-C I: 8, 12 or 24 weeks) and dependent on time to first undetectable HCV RNA using Roche Taqman HCV RNA testing. The primary efficacy endpoint was SVR12 by intention-to-treat. Logistic regression analyses were used to identify predictors of SVR.

Results: A total of 82 participants (62% HIV-positive) were enrolled in ATAHC II (treated, n=52) and 14 (79% HIV-positive) in DARE-C I. The predominant modes of HCV acquisition were injecting drug use (ATAHC II: 55%, DARE-C I: 36%) and sexual intercourse with a partner of the same sex (ATAHC II 39%, DARE-C I 64%). SVR12 was 71% in both ATAHC II (37/52) and DARE-C I (10/14) with 56% in ATAHC II receiving shortened therapy (8 or 16 weeks). SVR was associated with a rapid virological response (odds ratio 10.80; p=0.001).

Conclusions: The majority of participants were able to receive short duration response-guided therapy with pegylated-interferon alfa-2a and ribavirin. Response-guided therapy for recent hepatitis C infection could be considered in the absence of available interferon-free therapies.

Registration: ClinicalTrials.gov registry (ATAHC II: NCT01336010; DARE‑C I: NCT01743521).

Page 72 of 309 Introduction The management of recent (acute or early chronic) hepatitis C virus (HCV) infection is not standardised with uncertainty regarding the optimal regimen and treatment duration (298), particularly as the therapeutic landscape changes with the advent of interferon-free direct-acting antiviral (DAA) therapy (91, 103, 140).

Enhanced responsiveness with interferon-based therapy in recent HCV means that treatment duration can be shortened (298). Previous studies have demonstrated the efficacy of interferon mono-therapy (standard or pegylated [PEG-IFN]) for 4, 12 and 24 weeks (124, 128, 133, 137, 138) with previous international guidelines recommending 24 weeks of therapy (103, 301). Shorter treatment durations result in fewer adverse events, better quality of life, less frequent dose reductions and increased likelihood of optimal adherence (137).

As with chronic HCV, response-guided therapy may be appropriate. The Australian Trial in Acute Hepatitis C II (ATAHC II) evaluated the efficacy and safety of response-guided therapy with PEG-IFN alfa-2a and ribavirin for individuals with recent HCV infection. The Direct- acting Antiviral Based Therapy for Recently Acquired Hepatitis C (DARE-C I), a sub study of ATAHC II, assessed the efficacy and safety of response-guided therapy with PEG-IFN alfa-2a, ribavirin and telaprevir for individuals with recent genotype 1 HCV infection.

Page 73 of 309 Methods Australian Trial in Acute Hepatitis C II (ATAHC II) Study design ATAHC II was a prospective study of the natural history and treatment outcomes of recent HCV infection (estimated duration of infection ≤18 months) following response-guided therapy with PEG-IFN alfa-2a (180mcg/week) and ribavirin (genotype 1: 1000mg/day if <75kg, 1200mg/day if ≥75kg; genotype 2/3: 800mg/day).

Enrolled participants were assessed for treatment eligibility. Participants who were eligible and consented for treatment were stratified by HIV status and estimated duration of infection at baseline. Participants with acute (estimated duration of infection ≤6 months) HCV mono- infection received PEG-IFN; participants with early chronic infection (estimated duration 6-18 months) and HIV co-infection received PEG-IFN and ribavirin. Treatment duration was dependent on time to first HCV RNA below the limit of detection using COBAS Taqman HCV RNA assay, version 2.0 (lower limit of quantitation [LLoQ], 25 IU/mL; lower limit of detection [LLoD] 15 IU/mL; Roche Diagnostics, Branchburg, NJ, USA). Participants who were ineligible or declined treatment were followed in the untreated arm.

Setting and participants Adults (age ≥16 years) with recent HCV were eligible for study inclusion. Participants were screened and enrolled between August 2011 and July 2014 through an Australian network of tertiary hospitals (n=6) and general practice/primary care clinics (n=1) with the last participant completing 12 weeks post treatment follow up in May 2015. Details regarding inclusion and exclusion criteria and study assessments are provided in the Supplementary Material.

Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C (DARE-C I) Study design A sub-study of ATAHC II, DARE-C I assessed the efficacy and safety of response-guided therapy with PEG-IFN alfa-2a (180mcg/week), weight-based ribavirin (1000mg/day if <75kg, 1200mg/day if ≥75kg) and telaprevir (1125mg twice daily or 1125 three times daily if receiving efavirenz) for individuals with recent genotype 1 HCV infection (estimated duration of infection 6-18 months). Treatment duration was dependent on time to first HCV RNA below the limit of detection using COBAS Taqman HCV RNA assay.

Page 74 of 309 Setting and participants Adults (age ≥18 years) with recent genotype 1 HCV infection, HCV RNA ≥10,000 IU/mL, and hepatitis B surface antigen negative were eligible for enrolment. Patients were screened and enrolled between April 2013 and May 2014 at two tertiary hospitals in the ATAHC II network. Details regarding inclusion and exclusion criteria and study assessments are provided in the Supplementary Material.

Table 2-1. Treatment allocation in ATAHC II and DARE C I

HCV RNA Treatment duration Study Treatment regimen BLoD (weeks) Week 2 8 PEG-IFN +/- RBV Week 4 16 PEG-IFN +/- RBV ATAHC II Week 6 24 PEG-IFN +/- RBV Week 8 32 (24 for G2/3) PEG-IFN +/- RBV Week 12 48 (24 for G2/3) PEG-IFN +/- RBV Week 2 8 PEG-IFN/RBV/TVR

DARE-C I Week 4 12 PEG-IFN/RBV/TVR PEG-IFN/RBV/TVR for 12 weeks Week 8 24 + PEG-IFN/RBV for 12 weeks Abbreviations: BLoD, below limit of detection; PEG-IFN, pegylated interferon alfa-2a; RBV, ribavirin; TVR, telaprevir

Study Definitions for ATAHC II and DARE-C I Recent HCV infection was defined as initial detection of serum anti-HCV antibody and/or HCV RNA within six months of enrolment and either (i) documented recent HCV seroconversion (anti-HCV antibody negative result in the 24 months prior to enrolment) or (ii) acute clinical hepatitis (jaundice or alanine aminotransferase [ALT] greater than 10 times the upper limit of normal [ULN]) within the previous 12 months with the exclusion of other causes of acute hepatitis (15), with estimated duration of infection less than 18 months at screening. The duration of HCV infection at screening and baseline was calculated from the estimated date of infection.

HCV virological suppression was defined as HCV RNA below the lower limit of detection. An end-of-treatment response (ETR) was defined as serum HCV RNA below the lower limit of detection at the end of treatment. HCV RNA recurrence was defined as detectable HCV RNA following HCV virological suppression. Participants with recurrence had HCV RNA sequencing performed on the first available detectable HCV RNA sample and the first available detectable HCV RNA sample indicating HCV RNA recurrence. HCV virological failure was

Page 75 of 309 defined as non-response (failure of virological suppression on-treatment with quantifiable HCV RNA at all time points between baseline and end of treatment), breakthrough (an increase from non-quantifiable to quantifiable HCV RNA or to at least 1 log10 above nadir while on treatment) or post-treatment relapse (the presence of quantifiable HCV RNA after an ETR, confirmed by homologous virus on sequencing of Core-E2 and/or NS5B regions as described previously) (302, 303). Reinfection was defined by the detection of infection with an HCV strain that was distinct from the primary infecting strain.

Loss of HIV virological control was defined as a confirmed HIV RNA of at least 400 copies/mL in an individual on cART. For further study definitions, see Supplementary Material.

Study Outcomes for ATAHC II and DARE-C I The primary efficacy endpoint was SVR12, defined as serum HCV RNA below the limit of detection at 12 weeks following end of treatment. Secondary virological endpoints included a rapid virological response (RVR, defined as serum HCV RNA below the LLoQ prior to or at week 4 of treatment), ETR and SVR24 (defined as serum HCV RNA below the limit of detection at 24 weeks following end of treatment. SVR12 results for participants with HCV genotype 1 in ATAHC II were compared with DARE-C I. SVR24 results for participants in ATAHC II were compared to the historical controls in ATAHC I (PEG-IFN +/- ribavirin for 24 weeks) (133).

Study oversight All study participants provided written informed consent before study procedures. The study protocols were approved by St Vincent’s Hospital, Sydney Human Research Ethics Committee (primary study committee), as well as local ethics committees at all study sites. The studies were conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines. The studies were registered with clinicaltrials.gov registry (ATAHC II: NCT01336010; DARE C I: NCT01743521).

Statistical analysis Evaluation of HCV treatment response was based on intention-to-treat (ITT) analyses that included all participants who received at least one dose of PEG-IFN. Additional per protocol analyses included all adherent individuals with follow-up virological data to week 12 post treatment.

For all endpoints, means and proportions with two-sided 95% confidence intervals (CI) were determined, and were unadjusted for multiple comparisons. Continuous variables were

Page 76 of 309 analysed using ANOVA methods or non-parametric equivalents. Binary endpoints were analysed using chi-square methods or logistic regression. A Cox proportional hazards model was used to assess factors associated with time to first HCV RNA below the limit of detection and logistic regression analyses were used to identify baseline and on-treatment predictors of HCV treatment response. Potential predictors were determined a priori and included participant, virological and on-treatment characteristics. The multivariate model for predictors of treatment response and HCV clearance were determined using a backwards stepwise approach, considering factors that were significant at the 0.2 level in univariate analysis. The final models included factors that remained significant at the 0.05 level. All p-values are two- sided. Analyses were performed using STATA version 14.0 (Stata Corporation, College Station, TX).

Page 77 of 309 Results ATAHC II Participant disposition and overview of the study population Between August 2011 and July 2014, 112 individuals were screened and 82 enrolled (Figure 2- 1). Participants were predominantly male (89%) with genotype 1 (51%) and genotype 3 (46%) infection. HIV co-infection was documented in 62%. Diagnosis of recent HCV occurred in the context of acute clinical hepatitis in 68% (n=56) and asymptomatic anti-HCV antibody seroconversion in 32% (n=26). In those with acute clinical hepatitis, a symptomatic seroconversion illness was reported in 43% (n=35, including 17 with jaundice) and ALT >400 IU/mL in 59% (n=48). The predominant modes of acquisition were injecting drug use (55%, n=45) and sexual intercourse with a partner of the same sex (39%, n=32; all were men-who- have-sex-with-men [MSM]). Other modes of acquisition included heterosexual contact (5%, n=4) and other forms of percutaneous exposure (1%, n=1). The enrolment characteristics of treated (n=52) and untreated (n=30) participants are shown in Table 2-2.

Seventy-six percent (n=62) of participants had ever injected drugs with current injecting drug use (within the last 6 months) reported by 56% (n=46). Among participants who reported injecting drug use ever, median age at first injecting was 27 years (IQR 21-34), with older age at first injecting in those with HIV (median age 29 vs 23 years; p=0.012). Of those reporting injecting drug use within the last 6 months (n=46), 65% had injected in the previous month with methamphetamine (83%), heroin (7%) and other opiates (10%) most often injected.

Participants with HIV (n=51) were older, more likely to have acquired HCV through sexual exposure (p=0.002), be in full or part-time employment (p=0.001) and have better social functioning (p=0.007) (Supplementary Table 2-1).

Thirty participants were enrolled in the untreated arm. The reasons for not receiving treatment were PEG-IFN and/or ribavirin ineligibility (63%, n=19), patient choice (40%, n=12), inability to attend study visits (10%, n=3) and needle phobia (3%, n=1). The percentage total is greater than 100%, as more than one reason was identified for three individuals. Participants in the untreated arm were more likely to be unemployed, previously incarcerated, have injected drugs and report psychiatric co-morbidity (Table 2-2).

As one participant with undetectable HCV RNA at screening was ineligible, the uptake of HCV treatment was 64% (52/81). In the untreated arm, spontaneous clearance was observed in 14% (4/29).

Page 78 of 309 A Screened, n=112

Exclusions, n=30 Ineligible (n=1) Patient consent withdrawn (n=2) Lost to follow up (n=13) Enrolled into another study1 (n=6) HCV RNA undetectable at screening and timepoint prior to baseline (n=7) Unwell post screening (n=1) Enrolled and assessed for treatment, n=82

Treated arm, n=52 Untreated arm, n=30 (HIV-positive, n=37) (HIV-positive, n=14)

Treatment allocation HCV RNA positive at screening, 8 weeks, n=13 n=29 16 weeks, n=16 HCV RNA negative at screening, 24 weeks, n=11 n=1 48 weeks, n=2 Non-responder2, n=7 Early treatment discontinuation3, n=3

B Screened, n=15

Exclusions, n=1

Treated arm, n=14 (HIV-positive, n=11)

Treatment allocation 8 weeks, n=7

12 weeks, n=3 24 weeks, n=2 Non-responder1, n=1 Early treatment discontinuation2, n=1

Figure 2-1. Patient disposition. (A) ATAHC II. (B) DARE-C I.

Page 79 of 309 1 Of those excluded and enrolled into another study (n=6), 5 were enrolled into DARE-C I.

2 ATAHC II non-responder: Participants receiving PEG-IFN with or without RBV who do not achieve an HCV RNA below the limit of detection (LLoD <15 IU/ml on Roche TaqMan) after 12 weeks of treatment

3 Early treatment discontinuation: Treatment ceased prior to allocation of treatment duration

4 DARE C I non-responder: Participants in whom therapy was terminated at week 4 due to HCV RNA >1000 IU/mL or week 8 due to detectable HCV RNA.

Page 80 of 309 Table 2-2, Part A. Participant characteristics – by study and treatment allocation

ATAHC II DARE-C I Enrolment characteristics Overall Treated Untreated Overall (n=82) (n=52) (n=30) (n=14) Age (years), mean (SD) 39 (10) 41 (10) 36 (9) 48 (11)

Male, n (%) 73 (89) 48 (92) 25 (83) 14 (100)

Weight (kg), mean (SD) 77 (13) 79 (13) 74 (12) 77 (13)

BMI (kg/m2), mean (SD) 25 (3) 25 (3) 24 (3) 24 (3)

Caucasian ethnicity, n (%) 67 (82) 44 (85) 23 (77) 14 (100) Higher education or qualification a, n 51 (62) 36 (69) 15 (50) 8 (57) (%) Full or part time employment, n (%) 40 (49) 33 (63) 7 (23) 7 (50)

Incarceration ever, n (%) 5 (6) 1 (2) 4 (13) 0

Social functioning score, median (IQR) 12 (7-17) 10 (6-14) 15 (10-19) 13 (9-16)

Psychiatric history, n (%) 46 (56) 23 (44) 23 (77) 10 (71)

Current major depression, n (%) 16 (20) 11 (21) 5 (17) 5 (36)

Injecting drug use, n (%)

Ever 62 (76) 34 (65) 28 (93) 6 (43)

Current b 46 (56) 22 (42) 24 (80) 3 (21)

Opioid substitution therapy, n (%)

Ever 10 (12) 7 (13) 3 (10) 1 (7)

Current 6 (7) 3 (6) 3 (10) 0

HIV infection, n (%) 51 (62) 37 (71) 14 (47) 11 (79) 610 512 723 464 CD4 count (106/L), median (IQR) (441-754) (399-692) (624-860) (376-626) HIV VL ≤50 copies/mL, n (%) 33 (65) c 21 (57) c 12 (86) 8 (73)

On cART, n (%) 46 (90) 32 (86) 14 (100) 11 (100) a Completed higher technical qualification, college or university degree b Current injecting drug use refers to use within 6 months of screening c Recent diagnosis of HIV infection (within 2 years of study enrolment), n=11 (22%), with 10 enrolled in the treated arm. HIV RNA <50 copies/mL at screening in only 18% (2/11) with a recent HIV diagnosis compared with 78% (31/40) in those without (p<0.001).

Abbreviations: BMI, body mass index; cART, combination antiretroviral therapy

Page 81 of 309 Table 2-2, Part B. HCV virological characteristics – by study and treatment allocation

ATAHC II DARE-C I Enrolment characteristics Overall Treated Untreated Overall (n=82) (n=52) (n=30) (n=14) Mode of HCV acquisition, n (%) Injecting drug use 45 (55) 22 (42) 23 (77) 5 (36) Sexual exposure – same sex 32 (39) 26 (50) 6 (20) 9 (64) Sexual exposure – opposite sex 4 (5) 3 (6) 1 (3) 0 Other 1 (1) 1 (2) 0 0 Acute HCV (<6 months) a, n (%) 20 (38) 40 (49) 19 (63) 0

Estimated duration of infection (weeks)

At screening, median (IQR) 26 (14-35) 28 (19-39) 18 (9-29) 32 (26–40)

At baseline, median (IQR) 36 (27-46) 37 (30-46) 32 (25-49) 41 (36-56)

Presentation of recent HCV, n (%)

Acute clinical illness – symptomatic 35 (43) 22 (42) 13 (43) 5 (36) Jaundice b 17 (49) 11 (50) 6 (46) 2 (40) Nausea/vomiting b 12 (34) 8 (32) 4 (31) 2 (40) Abdominal pain b 13 (37) 8 (32) 5 (38) 3 (60) Fever b 10 (29) 6 (27) 4 (31) 3 (60) Acute clinical illness - ALT >10x ULN 48 (59) 32 (62) 16 (53) 10 (71)

Asymptomatic seroconversion 26 (32) 15 (29) 11 (37) 2 (14)

ALT, median (IQR) 621 621 575 623 Peak ALT prior to enrolment (U/L) (218-1129) (232-1110) (169-1247) (156-925) At screening (U/L) 135 127 142 116 (81-354) (83-360) (70-349) (53–151) HCV RNA at screening (log10 IU/mL), 5.7 5.9 4.7 6.3 median (IQR) (4.6-6.5) (5.1-6.6) (2.9-5.9) (6.2-6.8) HCV RNA <400,000 IU/mL, n (%) 38 (46) 19 (37) 19 (63) 5 (36)

HCV genotype (and subtype), n (%) Genotype 1 42 (51) 28 (54) 14 (47) 14 (100) 1a 41 (98) 28 (100) 13 (93) 13 (93) 1b 1 (2) 0 1 (7) 1 (7) Genotype 2 1 (1) 1 (2) 0 - Genotype 3 38 (46) 22 (42) 16 (53) - Genotype 4 1 (1) 1 (2) 0 - a Acute HCV infection (duration of infection <6 months) at screening b Denominator = number of people with acute clinical (symptomatic) illness

Abbreviations: ALT, alanine aminotransferase; ULN, upper limit of normal

Page 82 of 309 Efficacy of response-guided pegylated interferon and ribavirin In the treated cohort, SVR12 by ITT was 71% (37/52; 95% CI 57%, 83%), with no difference by HIV status (HCV mono-infection, SVR12 73% [11/15]; HIV/HCV co-infection, SVR12 70% [26/37]; p=0.825) (Figure 2-2). Treatment discontinuation due to virological non- response or early treatment discontinuation (prior to duration allocation) occurred in 19% (n=10).

By treatment duration (n=42), SVR12 was 85% (11/13) in those receiving 8 weeks, 100% (16/16) in those receiving 16 weeks, 73% (8/11) in those receiving 24 weeks and 100% (2/2) in those receiving 48 weeks. The majority (n=29, 56%) achieved a rapid virological response and received shortened therapy (8 or 16 weeks). In those who achieved a rapid virological response, SVR12 was 93% (27/29).

SVR12 was lower in those with genotype 1 (61%; 17/28) as compared with other the HCV genotypes (genotype 2 100%, 1/1; genotype 3 82%, 18/22; genotype 4 100%, 1/1; genotype 1 versus non- genotype 1, p=0.073). In those receiving 8 weeks, SVR12 was 86% in genotype 1 (6/7) and 83% in genotype 3 (5/6). In those receiving 16 weeks, SVR12 was 100% in genotype 1 (8/8), 100% in genotype 3 (7/7) and 100% in genotype 4 (1/1). In those receiving 24 weeks, SVR12 was 25% in genotype 1 (1/4), 100% in genotype 2 (1/1) and 100% in genotype 3 (6/6). In those receiving 48 weeks, SVR12 was 100% in genotype 1 (2/2). SVR12 by per-protocol analysis was 76% (37/49). HCV RNA was below the LLoQ in 27%, 56%, 69%, 77% and 83% at weeks 2, 4, 6, 8 and end of treatment, respectively.

SVR24 by ITT was 69% (34/49). Three participants with undetectable HCV RNA at SVR12 did not reach the SVR24 time point due to study closure. Efficacy data from ATAHC II was compared to historical data from ATAHC I (133). In those with HCV mono-infection, SVR24 by ITT in ATAHC II (73%) was higher, though not statistically different, when compared with ATAHC I (55%; 24 weeks PEG-IFN) (risk difference 0.18, 95% CI -0.07, 0.43; p=0.200). In those with HIV/HCV co-infection, SVR24 by ITT in ATAHC II (68%) was similar to ATAHC I (74%; 24 weeks PEG-IFN and ribavirin) (risk difference -0.07, 95% CI -0.28, 0.15; p=0.543).

Page 83 of 309 100 Total study population HCV mono-infection HIV/HCV co-infection

80

60

40 Proportionof participants (% )

20

43/52 13/15 30/37 8/19 37/52 11/15 26/37 3/14 34/49 11/15 23/34 0 ETR SVR12 SVR24

100 Total study population HCV mono-infection HIV/HCV co-infection

80

60

40 Proportionof participants (% )

20

12/14 3/3 9/11 8/19 10/14 2/3 8/11 3/14 9/14 2/3 7/11 0 ETR SVR12 SVR24

Figure 2-3. Primary and secondary efficacy endpoints by study (intention-to-treat population).

(A) ATAHC II – response-guided PEG-IFN and ribavirin.

(B) DARE C-I – response-guided PEG-IFN, ribavirin and telaprevir.

*Three participants with undetectable HCV RNA at SVR12 did not reach the SVR24 time point due to study closure. Abbreviations: ETR, end of treatment response; ITT, intention-to-treat; SVR, sustained virological response

Page 84 of 309 Virological Failure, Relapse and Reinfection Treatment discontinuation was noted in 19% due to virological non-response (n=6), medical contraindication to treatment continuation (n=1), and clinician decision (n=3) (including one participant with HCV RNA <15 IU/mL at baseline and HCV RNA undetectable at week 2). No on-treatment virological breakthrough was noted.

Virological suppression on treatment was documented in 83% (43/52) with recurrent HCV viraemia in 21% (9/43); relapse was demonstrated in six (with recurrence of viraemia at 12 and 24 weeks post treatment in five and one individuals, respectively) and reinfection in three participants (with recurrence of viraemia at one and two years post treatment in one and two individuals, respectively) (Figure 2-4). In the treated cohort at risk for HCV reinfection, reinfection incidence was 7.5 per 100 person years (py) (95% CI 1.6, 20.4).

Treatment adherence Adherence to therapy was high with PEG-IFN 80/80 (≥80% of doses for ≥80% of treatment period) and 100/100 (100% of doses for 100% of treatment period) adherence 100% and 98%, respectively (mean on-treatment PEG-IFN adherence 99.96% [SD 0.28%]) and ribavirin 80/80 and 100/100 adherence 94% and 67%, respectively (mean on-treatment ribavirin adherence 95.25% [SD 18.65%]). HIV/HCV co-infected participants were more likely to be ribavirin adherent than HCV mono-infected participants (ribavirin 80/80 100% vs 79%, p=0.004; ribavirin 100/100 76% vs 43%, p=0.045). PEG-IFN 80/80 adherence was better with response guided therapy in ATAHC II as compared with the prior standard 24 week duration in ATAHC I (100% vs 82%; p=0.001).

Factors associated with time to first HCV RNA below the limit of detection and SVR A Cox proportional hazards model was used to assess factors associated with time to first HCV RNA below the limit of detection. Higher baseline HCV RNA level (≥400,000 IU/mL) was negatively associated with time to HCV RNA below the limit of detection (HR 0.34, 95% CI 0.18, 0.64; p=0.001) (Supplementary Material).

Participant characteristics and on-treatment factors were evaluated as predictors of SVR with logistic regression analysis. Rapid virological response was the only factor associated with SVR (OR 10.80; 95% CI 2.51, 46.43; p=0.001) (Supplementary Material).

Page 85 of 309

8

6 (IU /m L) 10

4 HCV RNA log RNA HCV

2

0 SCR BL W2 W4 W6 W8 EOT PT W 12 PT W 24 PT W 48 PT W 72 PT W 96

8

6 (IU /m L) 10

4 HCV RNA log RNA HCV

2

0 SCR BL W2 W4 W6 W8 EOT PT W 12 PT W 24 PT W 48 PT W 72 PT W 96

Figure 2-4. ATAHC II: Viral kinetics and outcome in participants with recurrence of HCV viraemia post treatment

Panel A: Relapse (n=6). Panel B: Reinfection (n=3). Red lines indicate individuals with HIV/HCV co-infection. Black lines indicate individuals with HCV mono-infection. Note, in (A), two individuals demonstrated spontaneous clearance of HCV viraemia following relapse, and in (B), one individual demonstrated spontaneous clearance following reinfection.

Abbreviations: BL, baseline; EOT, end of treatment; PT, post treatment; SCR, screening; W, week.

Page 86 of 309 Safety The safety profile was consistent with the known side effects of PEG-IFN alfa-2a and ribavirin (Supplementary Material). At least one clinical adverse event was reported by 51 participants (98%). Most adverse events were of mild (75%) or moderate (24%) severity. PEG-IFN and ribavirin dose modification were required for toxicity in 6% (neutropenia, n=3) and 2% (anaemia, n=1), respectively. In those with HIV infection, median change in CD4 count at end of treatment was 119x106/L (IQR 47–234) with no loss of HIV virological control.

Three serious adverse events were reported: anxiety requiring hospitalisation in an individual with a psychiatric history (possibly related to study drug administration); attempted suicide in an individual with a psychiatric history (possibly related to study drug administration); and sialolithiasis requiring hospitalisation (unrelated to study drug administration). No decompensated liver disease or death occurred.

DARE-C I Patient disposition and overview of the study population Between April 2013 and May 2014, 14 participants (79% HIV-positive) were enrolled (Figure 2-1). Enrolment characteristics are shown in Table 2-2. Forty six percent (n=6) had ever injected drugs. Amphetamines were the most commonly injected drug ever (29%) and within the last 6 months (21%).

Efficacy of response-guided pegylated interferon, ribavirin and telaprevir SVR12 by ITT was 71% (10/14) (Figure 2-2). By treatment duration, SVR12 was 71% (5/7) in those receiving PEG/ ribavirin/telaprevir for 8 weeks, 100% (3/3) in those receiving PEG/ ribavirin/telaprevir for 12 weeks and 100% (2/2) in those receiving PEG/ ribavirin/telaprevir for 12 weeks + PEG/ ribavirin for 12 weeks (24 weeks). In those with HIV, SVR12 was 73% (8/11). Rapid virological suppression was demonstrated in the majority with HCV RNA below the LLoQ in 36%, 50%, 57%, 71%, 86%, 86% and 86% at weeks 1, 2, 3, 4, 6, 8 and end of treatment, respectively. There was no difference in SVR12 ITT between genotype 1-infected participants in ATAHC II and DARE C I (61% vs 71%; risk difference -0.11, 95% CI -0.41, 0.19; p=0.494).

Page 87 of 309 Virological Failure, Relapse and Reinfection Treatment failure was observed in 29%: 7% (n=1) early treatment discontinuation (day 3), 7% (n=1) non-response, 14% (n=2) relapse. Reinfection was documented in one HIV-positive male participant with recurrence of HCV viraemia between post-treatment week 12 and post- treatment week 24. High-risk sexual behaviour was described with no history of injecting drug use. The combined reinfection incidence in treated HIV-positive participants in ATAHC II and DARE-C I was 11.8 per 100 py (95% CI 3.3, 27.5).

Treatment adherence As with ATAHC II, adherence was high with PEG-IFN 100/100 adherence 100% and ribavirin 80/80 and 100/100 adherence 100% and 50%, respectively (mean on-treatment ribavirin adherence 98.9% [SD 1.6%]). Telaprevir 80/80 and 100/100 adherence were 100% and 64% (mean on-treatment telaprevir adherence 98.9% [SD 1.7%]).

Safety Multiple adverse events were documented in all participants with the most common being fatigue (73%) and rash (50%) (Supplementary Material). Three serious adverse events were reported: skin cancer (squamous cell carcinoma/basal cell carcinoma) requiring hospitalisation, axillary abscess requiring hospitalisation and appendicitis requiring hospitalisation. Adverse events requiring medical intervention, treatment cessation or dose modification occurred in 36% (n=5), with dose reduction of PEG-IFN and ribavirin in one (7%) and three (21%) individuals, respectively.

The addition of telaprevir was associated with excess haematological toxicity. Mean decrease in haemoglobin at end of treatment was 33 g/L (SD 18) in participants receiving PEG-IFN, ribavirin and telaprevir as compared with 20 g/L (SD 15) in participants receiving PEG-IFN and ribavirin (p=0.007). Anaemia (haemoglobin less than 100 g/L) developed on treatment in 5 participants (36%) receiving PEG-IFN, ribavirin and telaprevir as compared with 3 (6%) receiving PEG-IFN and ribavirin (p=0.008).

Page 88 of 309 Discussion Within ATAHC II and DARE-C I, the majority of participants with recent HCV infection were able to receive short duration (8-16 weeks) response-guided therapy, with the overall SVR similar to that observed with previously recommended 24 week regimens (133). The recent development of highly curative and safe interferon-free DAA regimens for chronic HCV infection, with treatment duration generally 12 weeks (229, 237, 238, 245, 304-306), offers significant promise. However, due to high drug pricing, access to interferon-free DAA therapy is restricted, even in high-income settings: by and within countries, by fibrosis stage and by former or current substance misuse (140, 307). While many individuals with diagnosed recent HCV infection are keen for treatment (133), access to interferon-free therapy will be denied for most due to mild fibrosis and/or recent drug use. This study demonstrates that recent HCV infection can be effectively and safely treated with short course PEG-IFN and ribavirin; a response-guided strategy could be considered for motivated individuals wishing to trial therapy at this initial assessment, with treatment cessation at week four if HCV RNA remains detectable. Population-level HCV treatment-as-prevention (TasP) strategies will be enhanced by early detection and increased HCV treatment uptake for those with recent HCV infection (75).

Very limited evidence exists for the use of DAAs in recent HCV infection (308). Telaprevir, in combination with PEG-IFN and ribavirin, demonstrated improved efficacy in chronic genotype 1 HCV as compared with PEG-IFN and ribavirin (202, 203). In the DARE-C I study, response- guided therapy with PEG-IFN, ribavirin and telaprevir was effective in the majority (regardless of HIV co-infection) although similar to that observed with PEG-IFN and ribavirin alone. Despite the short treatment duration, the side-effect profile, drug-drug interactions and treatment complexity seen with this regimen indicates that the addition of telaprevir offers no significant benefit.

Over the last decade, increasing HCV transmission has been observed in HIV-positive men- who-have-sex-with-men, largely associated with sexual and non- injecting drug use behaviour (57, 58, 67-69, 71, 309). In comparison with ATAHC I (133), a greater proportion of participants in ATAHC II and DARE-C I were HIV-positive while the proportion reporting injecting drug use remained the same, highlighting the changing patterns of HCV transmission in Australia. Reinfection rates following treatment of acute or recent HCV infection in this population are varied with very high incidence reported in some cohorts (269, 310, 311). In a recent meta-analysis of late viral recurrence following treatment for acute or chronic HCV infection, the incidence of reinfection following SVR in those with HIV was 3.2 per 100 py (95% CI 0.1, 12.3) (268). Multiple reinfections in individuals with ongoing high risk behaviour

Page 89 of 309 emphasise the need for continued surveillance and prevention strategies (270, 311). Despite concerns regarding adherence and reinfection, HCV treatment with interferon-containing and interferon-free regimens is feasible and successful in those populations considered to be “high- risk”, including people who inject drugs and people receiving opioid substitution therapy (OST) (246, 251, 312). As such, HCV treatment should not be delayed, but rather, should occur in concert with education and harm minimisation.

Limitations of these studies are noted. Although ATAHC II is one of the larger studies in recent HCV infection, the sample size means that Cox proportional hazards and logistic regression analyses were limited to assessment of key virological and treatment factors. DARE-C I was designed as a proof-of-concept study to determine the feasibility of this strategy, and despite the small enrolled population, the tolerability of the regimen was poor.

With advances in HCV therapeutics, management strategies for recent HCV infection will evolve rapidly over the next few years. With interferon-free DAA therapy now the standard-of- care for chronic HCV infection, the “efficacy advantage” of early treatment in recent HCV infection may be reduced (and possibly eliminated) (140). The paradigm of shortened therapy in recent HCV infection using interferon-free DAA combinations remains uncertain and requires evaluation.

Page 90 of 309 Supplementary Material

Methods

ATAHC II

Inclusion and exclusion criteria

Adults (age ≥16 years) with recent HCV infection were eligible for study inclusion. Participants with detectable HCV RNA at screening were assessed for treatment eligibility.

Exclusion criteria for enrolment in the treatment arm included: age 16-18 years; pregnant women or male partners of pregnant women; breast feeding; systemic anti-viral, anti-neoplastic or immunomodulatory therapy ≤6 months prior to first dose of study drug; any investigational drug ≤6 weeks prior to first dose of study drug; positive anti-HAV IgM Ab or anti-HBc IgM Ab at screening; alternative aetiology of chronic liver disease; decompensated liver disease; active thyroid disease; severe retinopathy; severe seizure disorder; immunologically mediated disease, chronic pulmonary disease with functional limitation, severe cardiac disease, organ transplantation (apart from corneal, skin or hair graft), malignancy, or other severe illness (including psychiatric) which would make the participant unsuitable; and the following lab values at screening: neutrophil count <1500 cells/mm3, platelet count <90,000 cells/mm3, creatinine >1.5 times the upper limit of normal, haemoglobin <12 g/dL in women or <13 g/dL in men. Heavy alcohol intake and active illicit drug use were not exclusion criteria. A drug and alcohol assessment was performed at screening to determine treatment suitability.

Study assessments

In the treated arm, study visits were undertaken at baseline, day 1 and weeks 2, 4, 6, 8, 12, 16, 20 and 24, depending on treatment duration, and post-treatment weeks 12, 24, 48 and 72, until the individual completed study follow-up or the study closed (May 2015). In the untreated arm, study visits were undertaken at baseline and weeks 4, 8, 12, 24, 48, 72 and 96, until the individual completed study follow-up or the study closed (May 2015). The presence of HCV RNA was assessed at all scheduled study visits. HCV GT was assessed at screening. Adverse events were recorded on all treated participants from screening until week 12 post treatment. Questionnaires were administered at screening and every 12 weeks through follow-up to obtain information on illicit drug use, social functioning (Opiate Treatment Index Social Functioning Scale (313)) and psychological parameters (Mini-International Neuropsychiatric Interview (314) and the Depression Anxiety Stress Scale (315)). Adherence to therapy was assessed at clinical review and by self-reported questionnaire.

Page 91 of 309 DARE-C I

Inclusion and exclusion criteria

Adults (age ≥18 years) with recent genotype 1 HCV infection, HCV RNA ≥10,000 IU/mL at screening and baseline and hepatitis B sAg negative were eligible for enrolment and treatment commencement.

The following additional inclusion criteria were required for HIV-positive individuals: 1. HIV >6 months duration, 2. CD4 count >200 cells/mm3 and HIV viral load <50 copies/ml on stable combination antiretroviral therapy (cART) or 3. CD4 count ≥500 cells/mm3 and HIV viral load <100,000 copies/mL not on cART. The following antiretroviral agents were permitted: tenofovir, lamivudine, emtricitabine, efavirenz, abacavir, raltegravir, etravirine, rilpivirine and ritonavir-boosted atazanavir.

Exclusion criteria were the same as ATAHC II and in addition included: infection with non-GT 1 HCV; injecting drug use (IDU) within the previous 4 weeks; poorly controlled diabetes mellitus (haemoglobin A1c ≥8.5%); prior treatment with HCV protease or polymerase inhibitors; congenital QT prolongation or family history of congenital QT prolongation or sudden death; pancreatitis; haemophilia or other bleeding disorder; serious bacterial or fungal infection; and the following lab values at screening: potassium <3.5 mmol/L, calculated creatinine clearance <50mL/min.

Study assessments

Study visits were undertaken at baseline, day 1, weeks 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24, depending on treatment duration, and post-treatment weeks 12, 24, 48 and 72. The presence of HCV RNA was assessed at all scheduled study visits. HCV genotype was assessed at screening. Adverse events were collected from screening until week 12 post treatment. The same questionnaires as in ATAHC II were administered at screening and every 12 weeks through follow-up evaluation (313-315).

Page 92 of 309 Study definitions for ATAHC II and DARE C I

Recent HCV infection was defined as initial detection of serum anti-HCV antibody and/or HCV RNA within six months of enrolment and either (i) documented recent HCV seroconversion (anti-HCV antibody negative result in the 24 months prior to enrolment) or (ii) acute clinical hepatitis (jaundice or alanine aminotransferase [ALT] greater than 10 times the upper limit of normal [ULN]) within the previous 12 months with the exclusion of other causes of acute hepatitis (15), with estimated duration of infection less than 18 months at screening.

The presentation of recent HCV infection at the time of diagnosis was classified as either acute clinical or asymptomatic infection. Acute clinical infection included participants with a documented clinical history of symptomatic seroconversion illness and those without clinical symptoms but with a documented peak ALT > 400 U/L at or before the time of diagnosis. Asymptomatic infection included participants with anti-HCV Ab seroconversion but no acute clinical symptoms or documented peak ALT > 400 U/L. The estimated date of clinical infection was calculated as six weeks before onset of seroconversion illness or six weeks before the first ALT >400 U/L. The estimated date of asymptomatic infection was calculated as the midpoint between the last negative anti-HCV antibody and the first positive anti-HCV antibody. For participants who were anti-HCV antibody negative and HCV-RNA positive at screening, the estimated date of infection was six weeks before enrolment, regardless of symptom status.

On-treatment adherence was calculated for each individually (PEG-IFN, ribavirin and telaprevir) by subtracting the number of missed doses from the total number of doses prescribed for therapy duration and dividing by the total number of doses prescribed for therapy duration. By pill count and self-reported questionnaire, compliance with each medication was individually calculated at the 80/80 and 100/100 adherence levels, defined as receipt of ≥80% or 100% of scheduled doses for ≥80% or 100% of the scheduled treatment period, respectively.

Statistical analysis

For all endpoints, means and proportions with two-sided 95% confidence intervals (CI) were determined, and were unadjusted for multiple comparisons. Continuous variables were analysed using ANOVA methods or non-parametric equivalents, as appropriate. Binary endpoints were analysed using chi-square methods or logistic regression. A Cox proportional hazards model was used to assess factors associated with time to first HCV RNA below the limit of detection and logistic regression analyses were used to identify baseline and on- treatment predictors of HCV treatment response. During study conception, potential predictors were considered a priori and included participant, virological and treatment characteristics including sex, age, weight, education, employment, accommodation, social functioning, opioid

Page 93 of 309 substitution therapy, mental health status (depression and suicidality, based on the Mini- International Neuropsychiatric Interview(314)), ethnicity, IDU characteristics, alcohol consumption, estimated duration of HCV infection, presentation (acute clinical, asymptomatic), peak and baseline ALT, baseline HCV RNA, and HCV genotype. However, given the final sample size (316), potential predictors included in the analysis were limited to key clinical and virological variables: sex, social functioning, HIV co-infection, IDU characteristics, presentation (acute clinical, asymptomatic), peak ALT, baseline (and on-treatment) HCV RNA, and HCV genotype (Supplementary Tables 2-2 and 2-3), in order to minimise the risk of both Type I and II error. The ‘rule of thumb’ that logistic and Cox models should be used with a minimum of 10 events per predictor variable may be overly conservative; depending on the scenario, five to nine events per predictor variable may be reasonable (316). Social functioning was calculated using a validated scale from the Opiate Treatment Index (313) that addresses employment, residential stability, interpersonal conflict, social support, and the role of drug use in the participant’s social networks. A higher value indicates poorer functioning (range: 0– 48). The multivariate model for predictors of treatment response and HCV clearance were determined using a backwards stepwise approach, considering factors that were significant at the 0.2 level in univariate analysis. The final models included only factors that remained significant at the 0.05 level. All p-values are two-sided. All analyses were performed using STATA version 13.0 (Stata Corporation, College Station, TX).

Page 94 of 309 Rationale for response-guided interferon-based therapy allocation Individualisation of interferon-based treatment related to baseline HCV RNA and early on- treatment virological response is accepted in the management of chronic HCV infection (317- 323). In the INDIV-2 study, Sarrazin et al examined the efficacy of individualised therapy with PEG-IFN and ribavirin in 398 treatment-naïve individuals with HCV genotype 1 infection. Participants were allocated to five different treatment durations (ranging between 24 and 72 weeks), based on baseline HCV RNA and time to undetectable HCV RNA (measured at weeks 4, 6, 8, 12, 24 and 30) (323). SVR among those receiving individualised therapy (n=398) was compared to a cohort of individuals with HCV genotype 1 infection who received standard-of- care, PEG-IFN and ribavirin for 48 weeks (n=225). Individualised (response-guided) therapy was non-inferior to standard therapy (individualized treatment, SVR 55%; standard treatment, SVR 48%; p<0.001 for non-inferiority). Participants with an RVR who received 24 or 30 weeks of interferon-based therapy achieved comparatively high SVR (86-88%). With this individualised (response-guided) approach, treatment duration was able to be reduced by 50% in comparison with standard-of-care in those with favourable virological characteristics.

Enhanced responsiveness with interferon-based therapy in recent HCV allowed treatment duration to be shortened in comparison with chronic infection (298). Studies in recent HCV infection demonstrated the efficacy of interferon mono-therapy (standard or pegylated [PEG- IFN]) for 4, 12 and 24 weeks (124, 128, 133, 137, 138), with previous international guidelines recommending 16-24 weeks of inteferon-based therapy (103, 301). As response-guided interferon-based therapy utilising shorter treatment durations was successful in chronic HCV, this strategy was examined in recent infection (Table 2-1).

Table 2-3. Treatment allocation in ATAHC II and DARE C I

HCV RNA Treatment duration Study Treatment regimen BLoD (weeks) Week 2 8 PEG-IFN +/- RBV Week 4 16 PEG-IFN +/- RBV ATAHC II Week 6 24 PEG-IFN +/- RBV Week 8 32 (24 for G2/3) PEG-IFN +/- RBV Week 12 48 (24 for G2/3) PEG-IFN +/- RBV Week 2 8 PEG-IFN/RBV/TVR

DARE-C I Week 4 12 PEG-IFN/RBV/TVR PEG-IFN/RBV/TVR for 12 weeks Week 8 24 + PEG-IFN/RBV for 12 weeks Abbreviations: BLoD, below limit of detection; PEG-IFN, pegylated interferon alfa-2a; RBV, ribavirin; TVR, telaprevir

Page 95 of 309 Tables Supplementary Table 2-1. ATAHC II: Participant enrolment characteristics, by HIV co- infection

HCV HCV/HIV Enrolment characteristics mono-infection co-infection P (n=31) (n=51) Age (years), mean (SD) 36 (9) 41 (8) 0.010 Male, n (%) 22 (71) 51 (100) <0.001 Weight (kg), mean (SD) 77 (17) 77 (10) 0.983 BMI (kg/m2), mean (SD) 25 (4) 25 (3) 0.311 Caucasian ethnicity, n (%) 25 (81) 42 (82) 0.846 Higher education or qualification a, n (%) 15 (48) 36 (71) 0.044 Full or part time employment 8 (24) 32 (63) 0.001 Prison/juvenile justice centre ever, n (%) 4 (13) 1 (2) 0.047 Social functioning score, median (IQR) 15 (9-21) 10 (6-14) 0.007 Current major depression, n (%) 8 (26) 8 (16) 0.262 Injecting drug use ever, n (%) Ever 26 (84) 36 (71) 0.220 Current b 19 (61) 27 (53) 0.209 In those reporting injecting drug use: Age at first injecting, median (IQR) 23 (18 – 30) 29 (25-38) 0.012 Last injected within last month, n (%) 14 (54) 16 (44) Last injected between 1-6 months ago, n (%) 5 (19) 11 (31) Last injected >6 months ago, n (%) 7 (27) 9 (25) Drug injected most in last month, n (%) Amphetamines 9 (64) 16 (100) 0.024 Heroin 2 (14) 0 Other opiates 3 (21) 0 Opioid substitution therapy, n (%) Ever 6 (19) 4 (8) 0.131 Current 6 (19) 0 0.002 Estimated duration of infection (weeks) At screening, median (IQR) 26 (11-33) 24 (14-40) 0.899 At baseline, median (IQR) 37 (27-43) 33 (27-50) 0.899 Acute clinical illness - jaundice +/- ALT >10x ULN 18 (58) 34 (67) 0.950 Asymptomatic seroconversion 13 (42) 17 (33) Mode of HCV acquisition, n (%) Injecting drug use 25 (81) 20 (39) Sexual exposure - same sex 3 (9) 29 (57) 0.002 Sexual exposure – opposite sex 2 (6) 2 (4) Other 1 (3) 0 a Completed higher technical qualification, college or university degree b Current injecting drug use refers to use within 6 months of screening Abbreviations: BMI, body mass index; cART, combination antiretroviral therapy

Page 96 of 309 Supplementary Table 2-2. ATAHC II: Factors associated with time to first HCV RNA below the limit of detection - Cox proportional hazards analysis

Variable HR 95% CI P Sex Male 1.00 - - Female 3.27 0.91, 11.73 0.069 Social functioning score <6 1.00 - - 7-12 0.75 0.36, 1.55 0.435 >12 1.11 0.54, 2.3 0.777 Injecting drug use ever No 1.00 - - Yes 0.85 0.44, 1.64 0.627 HIV co-infection No 1.00 - - Yes 0.65 0.34, 1.25 0.195 Presentation of acute HCV Asymptomatic seroconversion 1.00 - - Acute clinical 1.87 0.93, 3.72 0.080 Peak ALT ≤400 U/L 1.00 - - >400 U/L 0.65 0.34, 1.24 0.189 HCV RNA at baseline <400,000 IU/mL 1.00 - - ≥400,000 IU/mL 0.34 0.18, 0.64 0.001 HCV genotype Genotype 1 1.00 - - Genotype 2 0.94 0.13, 7.06 0.956 Genotype 3 1.19 0.65, 2.22 0.562 Genotype 4 1.56 0.21, 11.72 0.666

Page 97 of 309 Supplementary Table 2-3. ATAHC II: Factors associated with SVR12 - logistic regression analysis

SVR No SVR Variable OR 95% CI P N=37 N=15 Sex Male 35 (95) 13 (87) 1.00 - - Female 2 (5) 2 (13) 0.37 0.05, 2.91 0.346 Social functioning score <6 13 (35) 4 (27) 1.00 - - 7-12 12 (32) 5 (33) 0.74 0.16, 3.41 0.698 >12 12 (32) 6 (40) 0.62 0.14, 2.72 0.523 Injecting drug use - ever No 11 (30) 6 (40) 1.00 - - Yes 25 (68) 9 (60) 0.66 0.19, 2.31 0.516 Injecting drug use - frequency Have not injected in past month 13 (35) 7 (47) 1.00 - - Have injected in past month 12 (32) 2 (13) 3.23 0.56, 18.71 0.191 Never injected 11 (30) 6 (40) 0.68 0.25, 3.82 0.985 HIV co-infection No 11 (30) 4 (27) 1.00 - - Yes 26 (70) 11 (73) 0.90 0.22, 3.30 0.825 Presentation of acute HCV Asymptomatic seroconversion 10 (27) 7 (47) 1.00 - - Acute clinical illness 27 (73) 8 (53) 2.36 0.68, 8.22 0.177 HCV genotype 1 No 20 (54) 4 (27) 1.00 - - Yes 17 (46) 11 (73) 0.31 0.08, 1.15 0.080 HCV RNA at baseline <400,000 IU/mL 18 (49) 4 (27) 1.00 - - ≥400,000 IU/mL 19 (51) 11 (73) 0.38 0.10, 1.43 0.153 Rapid virological response No 10 (27) 12 (80) 1.00 - - Yes 27 (73) 3 (20) 10.80 2.51, 46.43 0.001

Page 98 of 309 Supplementary Table 2-4. ATAHC II: Safety – Clinical adverse events and laboratory parameters

Treatment group (weeks) Clinical and lab adverse All treated 8 16 24 48 events n=52 a n=13 n=16 n=11 n=2 Any adverse event, n (%) 347 (100) 88 (25) 117 (34) 82 (24) 19 (6) Grade 3 or 4, n (%) 4 (1) 0 0 1 1 Serious adverse events, n (%) 3 (1)

Adverse events Common (>10%), n (%) Fatigue 31 (60) 9 (69) 4 (25) 9 (82) 1 (50) Insomnia 27(52) 6 (46) 9 (56) 6 (55) 2 (100) Headache 17 (33) 5 (38) 7 (44) 3 (27) 1 (50) Nausea 17 (33) 5 (38) 6 (38) 3 (27) 2 (100) Arthralgia 14 (27) 7 (54) 1 (6) 5 (45) 1 (50) Myalgia 14 (27) 4 (31) 3 (19) 4 (36) 1 (50) Diarrhoea 13 (25) 2 (15) 4 (25) 7 (64) 0 -like illness 9 (17) 2 (15) 4 (25) 1 (9) 0 Depressed mood 8 (15) 1 (8) 3 (19) 2 (18) 2 (100) Injection site erythema 8 (15) 1 (8) 0 5 (45) 1 (50) Irritability 8 (15) 2 (15) 3 (19) 1 (9) 1 (50) Decreased appetite 7 (13) 2 (15) 3 (19) 1 (9) 0 Lethargy 7 (13) 0 4 (25) 3 (27) 0 Injection site reaction 6 (12) 0 3 (19) 1 (9) 0 Pruritus 6 (12) 3 (23) 1 (6) 1 (9) 1 (50) Rash 6 (12) 3 (23) 0 1 (9) 0

Mean on-treatment nadir Hb 122 (14) 120 (14) 121 (12) 119 (12) 104 (16) (g/L), SD Decrease in Hb >30g/L, n (%) b 24 (46) 6 (46) 8 (50) 7 (64) 2 (100) Decrease in Hb <100g/L, n (%) 3 (6) 1 (8) 1 (6) 0 1 (50) Decrease in Hb <85 g/L, n (%) 0 0 0 0 0 Decrease in ANC ≤0.75, n (%) 13 (25) 1 (8) 5 (31) 4 (36) 1 (50) Decrease in ANC ≤0.5, n (%) 4 (8) 1 (8) 2 (13) 1 (9) 0 Decrease in plt <50, n (%) 0 0 0 0 0 a Includes individuals with no allocated treatment duration due to early treatment discontinuation and virological non-response b Decease in Hb >30g/L at any time between baseline and end of treatment

Abbreviations: ANC, absolute neutrophil count; Hb, haemoglobin; plt, platelet

Page 99 of 309 Supplementary Table 2-5. DARE-C I: Safety – Clinical adverse events and laboratory parameters

DARE-C I

Clinical and lab adverse events All treated n=14

Any adverse event, n (%) 103 (100)

Grade 3 or 4, n (%) 0

Serious adverse events, n (%) 3 (21) Adverse events Common (>10%), n (%) Fatigue 10 (71) Rash 7 (50) Headache 6 (43) Insomnia 6 (43) Pruritus 6 (43) Nausea 5 (36) Myalgia 4 (29) Abdominal pain 3 (21) Irritability 3 (21) Perianal pain or discomfort 3 (21) Decreased appetite 2 (14) Mean on-treatment nadir Hb (g/L), SD 105 (17)

Decrease in Hb >30g/L, n (%) a 12 (86)

Decrease in Hb <100g/L, n (%) 5 (36)

Decrease in Hb <85 g/L, n (%) 2 (14)

Decrease in ANC ≤0.75, n (%) 2 (14)

Decrease in ANC ≤0.5, n (%) 1 (7)

Decrease in plt <50, n (%) 0 a Decrease in Hb >30g/L at any time between baseline and end of treatment

Abbreviations: ANC, absolute neutrophil count; Hb, haemoglobin; plt, platelet

Page 100 of 309 Figures

A 8

6 (IU /m L) 10

4

HCV RNA log RNA HCV 2

LLoQ LLoD

0 SCR BL W2 W4 W6 W8 W 12 ETR SVR12 SVR24

B 8

6 (IU /m L) 10

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HCV RNA log RNA HCV 2

LLoQ LLoD

0 SCR BL W2 W4 W6 W8 W 12 ETR SVR12 SVR24

C 8

6 (IU /m L) 10

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HCV RNA log RNA HCV 2

LLoQ LLoD

0 SCR BL W2 W4 W6 W8 W 12 ETR SVR12 SVR24

Supplementary Figure 2-1. ATAHC II: Viral kinetics and outcome, by treatment allocation

Panel A: 8 and 16 weeks (n=29). Panel B: 24 and 48 weeks (n=13). Panel C: Non-response and early treatment discontinuation (n=10). Participants with HIV/HCV co-infection indicated in red and HCV mono-infection in black.

Abbreviations: BL, baseline; ETR, end of treatment; LLoD, lower limit of detection; LLoQ, lower limit of quantitation; SCR, screening; SVR, sustained virological response; W1-12, week 1-12.

Page 101 of 309 A 8

6 (IU /m L) 10

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LLoQ LLoD

0 SCR BL H4 D1 W1 W2 W3 W4 W6 ETR SVR12 SVR24

B 8

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LLoQ LLoD

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C 8

6 (IU /m L) 10

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HCV RNA log RNA HCV 2

LLoQ LLoD

0 SCR BL H4 D1 W1 W2 W3 W4 W6 W8 ETR SVR12 SVR24

Supplementary Figure 2-2. DARE-C I: Viral kinetics and outcome, by treatment allocation

Panel A: 8 weeks (n=7). Panel B: 12 and 24 weeks (n=5). Panel C: Non-response and early treatment discontinuation (n=2). Participants with HIV/HCV co-infection indicated in red and HCV mono-infection in black.

Abbreviations: BL, baseline; H4, hour 4; D1, day 1; ETR, end of treatment; LLoD, lower limit of detection; LLoQ, lower limit of quantitation; SCR, screening; SVR, sustained virological response; W1- 12, week 1-12.

Page 102 of 309 Chapter 3 The efficacy, safety and feasibility of ultra-short duration interferon- free therapy in recent HCV infection

Chapter Introduction and Objectives While interferon-based therapy has good efficacy in acute and recent HCV infection, the side effect profile limits implementation. As interferon-free direct-acting antiviral regimens are established as the standard-of-care for chronic HCV infection, their role and activity in recent HCV infection requires evaluation. Sofosbuvir and ribavirin was the first interferon-free regimen approved by the US Food and Drug Administration (FDA). Highlighting the pace of HCV therapeutic development in the last decade, sofosbuvir was discovered in 2007, commenced Phase I trials in 2010, was submitted to the US FDA in April 2013, received Breakthough Therapy Designation in October 2013 and was approved for use in December 2013 (with ribavirin for genotypes 2 and 3, and with pegylated-interferon and ribavirin for genotypes 1 and 4). Sofosbuvir and ribavirin for 12–24 weeks is safe and well tolerated in chronic HCV infection with efficacy dependent on genotype and disease stage.

Open to enrolment in 2014, the Direct-Acting Antiviral Based Therapy for Recent HCV II study (DARE-C II; 2014-2016) evaluated the feasibility, efficacy and safety of sofosbuvir and weight- based ribavirin for six weeks in individuals with recent HCV infection (estimated duration of infection <12 months). While six weeks of sofosbuvir and ribavirin was safe and well tolerated, efficacy was sub-optimal. Treatment efficacy was related to baseline HCV RNA and early on- treatment viral kinetics. DARE-C II confirmed the feasibility of ultra-short direct-acting antiviral therapy and informed future trial design in this population. Further research is needed to determine whether more potent interferon-free direct-acting antiviral regimens will allow treatment duration to be shortened in recent HCV infection.

The manuscript has been published in Hepatology.

Publication Martinello M, Gane E, Hellard M, Sasadeusz J, Shaw D, Petoumenos K, Applegate T, Grebely J, Maire L, Marks P, Dore GJ, Matthews GV. Sofosbuvir and ribavirin for six weeks is not effective among people with recent HCV infection: The DARE-C II study. Hepatology. 2016; doi: 10.1002/hep.28844

Page 103 of 309 Declaration I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Marianne Martinello

Viral Hepatitis Clinical Research Program Kirby Institute, UNSW Australia Wallace Wurth Building, Sydney NSW 2052 t: +61 413 276 968 e: [email protected]

Page 104 of 309 Co-authorship Acknowledgement In the case of Chapter Two, the nature and extent of my contribution to the work was the following:

Author name Contribution (%) Nature of contribution Conducted the data analysis, contributed to study design and data collection, and led the Marianne Martinello 51 development, writing and critical revision of the manuscript Contributed to study design, implementation, Edward Gane 3 data collection and critical revision of the manuscript Contributed to study design, implementation, Margaret Hellard 2 data collection and critical revision of the manuscript Contributed to study design, implementation, Joe Sasadeusz 2 data collection and critical revision of the manuscript Contributed to study design, implementation, David Shaw 2 data collection and critical revision of the manuscript Contributed to study design and statistical Kathy Petoumenos 2 analysis of data Contributed to study design and critical revision Tanya Applegate 2 of the manuscript Contributed to study design and critical revision Jason Grebely 2 of the manuscript Coordinated implementation of the study and Laurence Maire 2 data collection. Contributed to revision of the manuscript. Coordinated implementation of the study. Philippa Marks 2 Contributed to study design and revision of the manuscript. Contributed to study design, data collection, Gregory J Dore 15 interpretation of findings and critical revision of the manuscript Contributed to study design, data collection, Gail V Matthews 15 interpretation of findings and critical revision of the manuscript

Page 105 of 309 Sofosbuvir and ribavirin for six weeks is not effective among people with recent HCV infection: The DARE-C II study.

Marianne Martinello1, Edward Gane2, Margaret Hellard3, Joe Sasadeusz4, David Shaw5, Kathy Petoumenos1, Tanya Applegate1, Jason Grebely1, Laurence Maire1, Philippa Marks1, Gregory J Dore1, Gail V Matthews1

1. The Kirby Institute, UNSW Australia, Sydney, NSW, Australia 2. Auckland Hospital, Auckland, New Zealand 3. Burnet Institute, Melbourne, VIC, Australia 4. Royal Melbourne Hospital, Melbourne, VIC, Australia 5. Royal Adelaide Hospital, Adelaide, SA, Australia

Page 106 of 309 Abstract Background and Objectives: While interferon-based therapy has excellent efficacy in acute and recent hepatitis C virus (HCV) infection, the side effect profile limits implementation. Sofosbuvir and ribavirin for 12–24 weeks is safe and well tolerated in chronic HCV with efficacy dependent on genotype and disease stage. The aim of this study was to assess the efficacy of sofosbuvir and ribavirin for six weeks in individuals with recent HCV infection.

Methods: In this open-label study conducted in Australia and New Zealand, adults with recent HCV (duration of infection <12 months) received sofosbuvir 400mg daily and weight-based ribavirin (<75kg: 1000mg/day; ≥75kg: 1200mg/day) for six weeks. The primary efficacy endpoint was sustained virological response at post-treatment week 12 (SVR12).

Results: Nineteen participants commenced sofosbuvir and ribavirin (89% male, 74% HIV, 68% genotype 1a). The modes of acquisition were injecting drug use (53%) and sexual exposure in men-who-have-sex-with-men (47%). Twenty-one percent had a symptomatic seroconversion illness (n=4, including 2 with jaundice). At baseline, median HCV RNA was 5.4 log10 IU/mL (IQR 4.4-6.8) and median estimated duration of infection was 37 weeks (IQR 27-41). At end- of-treatment, HCV RNA was non-quantifiable in 89% (n=17). SVR4 and SVR12 were 42% (n=8) and 32% (n=6), respectively. Treatment failure was due to non-response (n=2), post- treatment relapse (n=9), reinfection (n=1) and loss to follow up (n=1). The regimen was well tolerated with minimal haematological toxicity. SVR12 was related to baseline HCV RNA (≤6 log10 IU/mL, p=0.018) and early on-treatment viral kinetics (HCV RNA below the level of quantitation at week 1, p=0.003).

Conclusions: Six weeks of sofosbuvir and ribavirin was safe and well tolerated, but efficacy was sub-optimal. Further research is needed to determine whether more potent interferon-free direct-acting antiviral regimens will allow treatment duration to be shortened in recent, predominantly asymptomatic, HCV infection.

Registration: ClinicalTrials.gov registry (NCT02156570).

Page 107 of 309 Introduction The management of recent hepatitis C virus (HCV) infection is not standardised, with uncertainty regarding the optimal regimen and treatment duration (298). As interferon-free direct-acting antiviral (DAA) regimens are established as the standard-of-care for chronic HCV infection (91, 103, 140), their role and activity in recent HCV infection requires evaluation. Most individuals with chronic HCV infection (>95%) will demonstrate a sustained virological response (SVR) following only eight to twelve weeks of DAA therapy (228, 230, 239, 324). Shorter treatment durations should aim to optimise adherence and enhance cost-effectiveness. With interferon-based regimens, 12 weeks treatment is sufficient to cure greater than 90% of individuals with acute HCV infection (298), as compared with 24-48 weeks treatment for chronic HCV infection. The efficacy of ultra-short duration interferon-free DAA therapy in individuals with acute and recent HCV infection is not known.

As individuals with diagnosed recent HCV infection are keen to consider treatment (133), this initial assessment may represent an ideal opportunity for therapeutic intervention, offering both individual and population level benefits (75). Successful treatment of acute HCV infection in people who inject drugs (PWID) and men-who-have-sex-with-men (MSM), when combined with implementation of harm reduction strategies should prevent transmission (Treatment-as- Prevention) and contribute towards HCV elimination at a population level (75, 300).

Sofosbuvir and ribavirin for 12-24 weeks have been evaluated as treatment for chronic genotype 1-4 HCV infection, with variable efficacy (high in genotype 2 [SVR12 88-97%], moderate in genotypes 1 [SVR12 68-85%] and 3 [SVR12 56-89%]) (243, 325, 326). The aim of this study was to assess the efficacy of sofosbuvir and ribavirin for six weeks in individuals with recent HCV infection (estimated duration of infection less than 12 months).

Page 108 of 309 Methods Study design DAA-based therapy for recent HCV II (DARE-C II) was a prospective, open-label multicenter pilot study in which adults with recent HCV infection received sofosbuvir 400mg daily and weight-based ribavirin (<75kg: 1000mg/day; ≥75kg: 1200mg/day) for six weeks. Recent HCV infection was defined as initial detection of serum anti-HCV antibody and/or HCV RNA within six months of enrolment and either (i) documented recent HCV seroconversion (anti-HCV antibody negative result in the 18 months prior to enrolment) or (ii) acute clinical hepatitis (jaundice or alanine aminotransferase [ALT] greater than 10 times the upper limit of normal [ULN]) within the previous 12 months with the exclusion of other causes of acute hepatitis (15), with calculated estimated duration of infection less than 12 months at screening.

The decision regarding HCV treatment suitability and timing of treatment initiation was made by the investigator on an individual basis at site level. Sites were instructed to observe participants for four to 12 weeks between screening and baseline (treatment day 1), providing an opportunity to assess for spontaneous clearance.

Study participants and setting Participants were enrolled between October 2014 and May 2015 through a network of tertiary hospitals in Australia (n=4) and New Zealand (n=1).

Adults (age ≥18 years) with recent HCV infection and HCV RNA ≥10,000 IU/mL at screening were eligible for study inclusion. Individuals with HIV co-infection were eligible provided: 1. HIV infection >6 months duration and 2. CD4 count >200 cells/mm3 and HIV viral load <50 copies/ml on stable combination antiretroviral therapy (cART) or 3. CD4 count ≥500 cells/mm3 and HIV viral load <100,000 copies/mL not on cART. The following antiretroviral agents were not permitted based on drug-drug interactions and potential adverse events: didanosine (327), zidovudine (328) and ritonavir-boosted tipranavir (210). Individuals with acute or chronic HBV co-infection were excluded. For the complete inclusion and exclusion criteria, see Supplementary Material.

Study definitions HCV virological suppression was defined as HCV RNA below the lower limit of quantitation (LLoQ) (target not detected [TND] or target detected, not quantifiable [TDnq]). An end-of- treatment response (ETR) was defined as serum HCV RNA below the LLoQ at the end of treatment. HCV virological failure was defined as non-response (failure of virological

Page 109 of 309 suppression on-treatment with quantifiable HCV RNA at all time points between baseline and end of treatment), breakthrough (an increase from non-quantifiable to quantifiable HCV RNA or to at least 1 log10 above nadir while on treatment) or post-treatment relapse (the presence of quantifiable HCV RNA after an ETR, confirmed as homologous virus on sequencing of Core- E2 and/or NS5B regions as described previously) (302, 303). Reinfection was defined by the presence of quantifiable HCV RNA after an ETR and detection of infection with an HCV strain that was distinct from the primary infecting strain (heterologous virus on sequencing of Core-E2 and/or NS5B regions). Loss of HIV virological control was defined as a confirmed HIV RNA of at least 400 copies/mL in individuals on cART. For further study definitions, see Supplementary Material.

Study assessments Study visits were undertaken at baseline (treatment day 1), treatment day 2 and treatment weeks 1, 2, 3, 4 and 6 and post-treatment weeks 4, 12 and 24. The presence of HCV RNA was assessed at all scheduled study visits using COBAS Taqman HCV RNA assay, version 2.0 (LLoQ, 25 IU/mL; lower limit of detection [LLoD] 15 IU/mL; Roche Diagnostics, Branchburg, NJ, USA), with centralised testing performed at SydPath (390 Victoria St, Darlinghurst, NSW, Australia). HCV genotype was assessed at screening and in the setting of HCV RNA recurrence. Participants with HCV RNA recurrence had HCV RNA sequencing of CoreE2 and/or NS5B regions performed on the first available detectable HCV RNA sample and the first available detectable HCV RNA sample indicating HCV RNA recurrence. All participants were genotyped for interferon-λ 3/4 (IFNL3/4) rs8099917 and rs12979860 single nucleotide polymorphisms (SNPs), as described previously (329, 330). Adverse events were recorded from screening until post-treatment week 12.

Behavioural questionnaires were administered at screening, baseline, end-of-treatment, post- treatment week 12 and post-treatment week 24. The behavioural questionnaire included sections on demographics (age, sex, ethnicity, education, main source of income and accommodation), opioid substitution treatment (including methadone and buprenorphine), and injecting drug use. At screening, injecting drug use history was collected for lifetime (ever), previous six months (current) and the previous month (recent). Recent (previous month) associated risk behaviours including use of a new sterile needle/syringe for all injections, needle/syringe borrowing and lending, and ancillary injecting equipment sharing were also collected. Follow-up information on injecting drug use and associated risk behaviours in the previous month were used for subsequent longitudinal analyses. Social functioning was assessed using the Opiate Treatment Index Social Functioning Scale (313).

Page 110 of 309 Study drug adherence was assessed by pill count and self-reported adherence questionnaires at treatment weeks 2, 4 and 6 (end-of-treatment). Plasma samples were taken and stored for therapeutic drug monitoring at baseline hour 4, treatment day 2 and treatment weeks 1, 2, 3, 4 and 6. Ribavirin plasma concentrations (mg/L) were quantified for all of these time points using a validated High-Performance Liquid Chromatography (HPLC) assay with UV detection (HPLC-UV; λ 235 nm) (331), with the following HPLC conditions: Atlantis C18 column (Waters, Milan, Italy), mobile phase 50 mM potassium phosphate buffer, pH 3.23 at 1 mL/min, isocratic mode, temperature 35ºC.

Study Outcomes The primary efficacy endpoint was SVR12, defined as serum HCV RNA below the LLoQ (target not detected [TND] or target detected, not quantifiable [TDnq]) at post-treatment week 12. Secondary virological endpoints included ETR, SVR4 (defined as serum HCV RNA below the LLoQ at post-treatment week 4) and SVR24 (defined as serum HCV RNA below the LLoQ at post-treatment week 24).

Study oversight All study participants provided written informed consent before study procedures. The study protocol was approved by St Vincent’s Hospital, Sydney Human Research Ethics Committee (primary study committee), as well as local ethics committees at all study sites. The study was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines. The study was registered with clinicaltrials.gov registry (NCT02156570).

Statistical analysis Primary efficacy and safety data were analysed based on intention-to-treat (ITT) population, including all participants who received at least one dose of therapy. Loss to follow-up was deemed treatment failure. Additional per-protocol efficacy analysis included all participants who completed the intended treatment course and who had follow-up virological data to post- treatment week 12.

Categorical parameters were summarised as number and proportion. Continuous variables were summarised by either mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. For all efficacy endpoints, means and proportions with two-sided 95% confidence intervals (CI) were determined. Categorical data was analysed using Fisher’s exact test. Continuous variables were analysed using the Mann-Whitney U test. The proportion of individuals engaging in injecting drug use and associated risk behaviours during treatment and

Page 111 of 309 follow-up were assessed up until post-treatment week 12. Changes in injecting behaviour between screening, end of treatment and post-treatment week 12 were compared using the McNemar test (exact binomial probability). All statistical tests were two-sided with a significance level of 0.05. Analysis was performed using STATA (version 14.0; StataCorp, College Station, TX).

Page 112 of 309 Results Participant disposition and overview of the study population Between October 2014 and May 2015, 26 individuals were screened and 19 enrolled (Figure 3-1). Participants were predominantly male (89%), with genotype 1 (68%) and genotype 3 (26%) infection (Table 3-1). HIV co-infection was documented in 74%. Sixty-eight percent (n=13) of participants had acute clinical hepatitis at the time of diagnosis and the remaining 32% (n=6) demonstrated asymptomatic anti-HCV antibody seroconversion. Twenty-one percent had a symptomatic seroconversion illness (n=4, including 2 with jaundice) and 63% (n=12) had an ALT greater than 400 U/L. The predominant modes of acquisition were injecting drug use (53%, n=10) and sexual exposure in men-who-have-sex-with-men (MSM) (47%, n=9). Median estimated duration of infection at screening and baseline were 32 (range 9–51) and 37 (range 12-55) weeks, respectively.

Screened, n=26

Exclusions, n=7 Ineligible (n=1) Patient consent withdrawn (n=1) HCV RNA <10,000 IU/mL (n=1) or undetectable (n=2) at screening Unwell post screening (n=1) Poor venous access (n=1)

Enrolled and assessed for treatment, n=19

Treated arm, n=19 (HIV-positive, n=14; HIV-negative, n=5)

Analysed as intention-to-treat population, n=19 Early treatment discontinuation and loss to follow up (week 2), n=1

Figure 3-1. DARE-C II: Patient disposition

Page 113 of 309 Table 3-1. DARE-C II: Participant demographic and clinical enrolment characteristics

Total study population Enrolment characteristics (n=19) Age (years), median (IQR) 41 (31-50) Male, n (%) 17 (89) Weight (kg), median (IQR) 76.0 (64.0-89.7) BMI (kg/m2), median (IQR) 22.6 (21.4-26.2) Caucasian ethnicity, n (%) 14 (74) Higher education or qualification a, n (%) 15 (79) Full or part time employment, n (%) 14 (74) Social functioning score, median (IQR) 14 (8-16) HIV infection, n (%) 14 (74) CD4 count (106/L), median (IQR) 598 (486-752) HIV VL ≤50 at screening, n (%) 11 (79) b On cART, n (%) 12 (86) c Mode of HCV acquisition, n (%) Injecting drug use 10 (53) Sexual exposure – MSM 9 (47) Estimated duration of infection (weeks) At screening, median (IQR) 32 (23-35) At baseline, median (IQR) 37 (27-41) Acute HCV c, n (%) 6 (32) Presentation of recent HCV, n (%) Acute clinical illness – symptomatic 4 (21) Jaundice 2 (11) Nausea/vomiting 3 (16) Abdominal pain 1 (5) Fever 1 (5) Acute clinical illness - ALT >10x ULN 12 (63) Asymptomatic seroconversion 6 (32) ALT (U/L), median (IQR) Peak ALT prior to enrolment 507 (228-959) ALT at screening 139 (78-323)

Log10 HCV RNA at baseline, median (IQR) 5.4 (4.4-6.8) HCV genotype (and subtype), n (%) 1a 13 (68) 2b 1 (5) 3a 4 (21) 3, unable to subtype 1 (5) IFNL3/4 (formerly IL28B) SNPs, n (%) rs12979860 CC 7 (37) rs8099917 TT 14 (74) Median liver stiffness measurement (Fibroscan®), kPa (IQR) 6.5 (5.3-7.4) a Completed higher technical qualification, college or university degree b HIV RNA >50 copies/mL in 1 HIV-positive participant on cART at screening (HIV RNA 149 copies/mL) c Acute HCV infection (duration of infection <24 weeks) at screening

Abbreviations: Combination antiretroviral therapy (cART), men-who-have-sex-with-men (MSM), SNP (single nucleotide polymorphism)

Page 114 of 309 Eighty-four percent (n=16) of participants had ever injected drugs with 58% (n=11) reporting injecting drug use within the last 6 months (Table 3-2). Among participants who reported injecting drug use ever, median age at first injecting was 28 years (IQR 19-42), with older age at first injecting in those with HIV co-infection (median age 30 [IQR 27-42] vs 19 [IQR 18-19]; p=0.049). Among those reporting recent injecting drug use at screening (n=6), the drugs most often injected were methamphetamine (83%) and heroin (17%).

Table 3-2. DARE-C II: History of substance use at enrolment

Total study population Substance use characteristics (n=19) Injecting drug use, n (%) Ever 16 (84) Current a 11 (58) In those reporting injecting drug use: Age at first injecting, median (IQR) 28 (19-42) Last injected within last month, n (%) 6 (38) Last injected between 1-6 months ago, n (%) 5 (31) Last injected >6 months ago, n (%) 5 (31) If injected in the previous 1 month, frequency (n, %): Daily 1 (17) More than weekly, not daily 1 (17) Less than weekly 4 (67) Drug injected most in last month, n (%) Amphetamines 5 (83) Heroin 1 (17) Opioid substitution therapy, n (%) Ever 1 (5) Current 1 (5) Alcohol use, n (%) None 3 (16) 1-2 standard drinks per week b 7 (37) >2 standard drinks per week b 9 (47) a Current injecting drug use refers to use within 6 months of screening b One standard drink equates to 10 grams of alcohol

Page 115 of 309 Efficacy By ITT analysis, SVR12 was achieved in 32% (6/19; 95% CI 13%, 57%) (Figure 3-2). In those with HIV co-infection, SVR4 and SVR12 were 36% (5/14) and 21% (3/14), respectively, as compared with 60% (3/5) and 60% (3/5), respectively, in those with HCV mono-infection (SVR4, p=0.603; SVR12, p=0.262) (Supplementary Material). By genotype, SVR4 and SVR12 were 31% (4/13) and 23% (3/13), respectively, in genotype 1, 100% (1/1) and 100% (1/1), respectively, in genotype 2 and 60% (3/5) and 60% (3/5), respectively, in genotype 3 (Supplementary Material). By per-protocol analysis, SVR12 was 33% (6/18; 95% CI 13%, 59%).

Virological suppression at end of treatment was documented in 89% (17/19) with similar results by HIV status (HCV mono-infection 100%, 5/5; HIV/HCV co-infection 86%, 12/14; p=1.000) (Figure 3-2). HCV RNA was below the LLoQ in 47%, 74% and 79% at weeks 1, 2 and 4 of treatment, respectively (Figure 3-3). A rapid biochemical response on treatment was observed in the majority (Supplementary Material); median ALT at baseline and end of treatment were 133 U/L (IQR 54-271) and 22 U/L (IQR 18-25), respectively (p<0.001).

SVR24 was 37% (7/19; 95% CI 16%, 62%) (Figure 3-2). SVR24 was confirmed in one HIV- positive genotype 3 male participant who had detectable HCV RNA at end-of-treatment (HCV RNA 31 IU/mL). He developed a clinical acute hepatitis at post-treatment week 4 (ALT 780 U/L; HCV RNA 136400 IU/mL), which resolved by post-treatment week 12 (ALT 25 U/L; HCV RNA 50 IU/mL). HCV RNA was not detected (TND) at post-treatment week 24 and confirmed at post-treatment weeks 28 and 48.

Page 116 of 309 100 Total study population HCV mono-infection 80 HIV/HCV co-infection

60

40

20 Proportionof participants (% )

17/19 5/5 12/14 8/19 3/5 5/14 6/19 3/5 3/14 7/19 3/5 4/14 0 ETR SVR4 SVR12 SVR24

Figure 3-2. DARE-C II: Primary and secondary efficacy endpoints (ITT population)

Abbreviations: ETR, end of treatment response; ITT, intention-to-treat; SVR, sustained virological response

100 HCV RNA TND HCV RNA TND and TDnq

80

60

** *

40

** Proportionof participants (% ) 20

0 D2 W1 W2 W4 EOT PT W4 PT W 12

Figure 3-3. DARE-C II: Proportion of participants with HCV RNA below the LLoQ (TND and TDnq, <15 IU/mL) on and post-treatment

** At week 1, 47% (n=9) of participants had HCV RNA TND or TDnq (<15 IU/mL) and 26% (n=5) had HCV RNA TND; 66% (n=6) and 80% (n=4), respectively, achieved SVR12 (HCV RNA TND or TDnq week 1, p=0.006; HCV RNA TND week 1, p=0.017). * At week 2, 47% (n=9) of participants had HCV RNA TND; of these, 56% (n=5) achieved SVR12 (p=0.057). Excluding those participants with loss to follow up (n=1) and reinfection (n=1), SVR12 was associated with HCV RNA TND at week 2 (p=0.035), but not HCV RNA TND or TDnq (p=0.102).

Abbreviations: BL, baseline; D2, day 2; EOT, end of treatment; LLoD, lower limit of detection; LLoQ, lower limit of quantitation; PT, post treatment; SCR, screening; SVR, sustained virological response; W1- 4, week 1-4

Page 117 of 309 Treatment efficacy was related to baseline HCV RNA (Figure 3-4) and early on-treatment viral kinetics (Figure 3-3). Median log10 HCV RNA at baseline and treatment day 2 were significantly lower in those achieving SVR12 (baseline: 4.47 IU/mL [IQR 3.48-4.87] vs 6.26 IU/mL [IQR 5.36-6.82], p=0.014; day 2: 3.08 IU/mL [IQR 2.06, 3.27] vs 4.85 IU/mL [IQR

4.07-5.39]; p=0.008). Fifty-five percent (6/11) of participants with baseline HCV RNA ≤6 log10

IU/mL achieved SVR12 compared to 0% (0/8) of those with baseline HCV RNA >6 log10 IU/mL (p=0.018). Sixty-seven percent (6/9) of participants with HCV RNA below the LLoQ (TND or TDnq [<15 IU/mL]) at week 1 achieved SVR12 as opposed to 0% (0/10) with quantifiable HCV RNA at week 1 (p=0.006) (Figure 3-3). There was no association between SVR12 and HCV RNA below the LLoQ at week 2 (p=0.114) or week 4 (p=0.515). Only two of the study participants were female, both of whom achieved SVR12 (p=0.088). Similarly, both participants with jaundice at diagnosis of acute HCV infection achieved SVR12 (p=0.088). SVR12 was not associated with any other baseline or on-treatment factors, including duration of infection, HIV infection, or rs8099917/rs12979860 SNP.

8

6 (IU/m L) 10

4

HCV RNAHCV log 2

0

Figure Non- SVR12 Reinfection LTFU Relapse legend response

Figure 3-4. DARE-C II: Treatment outcome by baseline HCV RNA

Note, 1. ETR documented in participant prior to LTFU and 2. SVR4 documented in participant prior to reinfection at post-treatment week 12.

Abbreviations: ETR, end-of-treatment response; LTFU, lost to follow up; SVR, sustained virological response

Page 118 of 309 Virological Failure, Relapse and Reinfection At post-treatment week 12, 13 (68%) of 19 had treatment failure. One participant discontinued after only two weeks treatment and was lost to follow up (LTFU, HCV RNA TND at end of treatment). The remaining 12 participants demonstrated virological failure: two non-response, nine post-treatment relapse and one reinfection. Both participants with virological non-response had high HCV RNA at baseline (>6.8 log10 IU/mL). Virological failure (excluding the two participants with reinfection and LTFU) was associated with baseline HCV RNA >6 log10 IU/mL (100% [8/8] vs 33% [3/9], p=0.009).

Reinfection was documented in an HIV-positive male participant with recurrence of HCV viraemia between post-treatment weeks four and 12, with a switch from genotype 3a at screening to genotype 1a at post-treatment week 12, confirmed on sequencing of CoreE2. Ongoing injection drug use and high-risk sexual behaviour were reported.

Treatment adherence Eighteen (95%) participants completed the scheduled six week treatment course. Adherence to therapy was high, by pill count and self-reported questionnaire. Sofosbuvir 90/90 and 100/100 adherence were 89% and 79%, respectively, with mean on-treatment sofosbuvir adherence 96.24% (SD 13.62%). Ribavirin 90/90 and 100/100 adherence were 89% and 84%, respectively, with mean on-treatment ribavirin adherence 98.18% (SD 4.73%) (Supplementary Material).

Consistent with self-reported adherence, when sampled, all participants had detectable ribavirin plasma concentrations on treatment. Median plasma ribavirin concentration at week 4 was 2.1 mg/L (range 1.00, 5.68) (Supplementary Material). Although median ribavirin plasma concentration was numerically lower in those who did not achieve SVR12, there was no significant association between SVR12 and plasma ribavirin concentration at week 4 (p=0.745) or week 6 (p=0.688), with a median week 4 ribavirin plasma concentration of 2.39 mg/L (IQR 1.00, 4.38) in participants achieving SVR12 as compared with 1.87 mg/L (IQR 1.35, 5.68) in participants with treatment failure. Both participants with virological non-response reported 100/100 adherence to sofosbuvir and ribavirin. At end of treatment, ribavirin plasma concentrations were 1.78 mg/L and 3.24 mg/L, respectively, with corresponding declines in haemoglobin of 22 and 24 g/L.

Page 119 of 309 Safety The safety profile was consistent with the reported side effects of sofosbuvir and ribavirin to date with no decompensated liver disease, death or treatment discontinuation due to adverse events (Table 3-3). One or more clinical adverse events were reported by 14 participants (74%), with most adverse events of mild (68%) or moderate (29%) severity. No ribavirin dose modification was required. In those with HIV infection, median change in CD4 count at end of treatment was 57x106/L (IQR -33–108) with no loss of HIV virological control. Two serious adverse events (SAE) were reported: acute kidney injury requiring hospitalisation; and Escherichia coli bloodstream infection (urinary source) requiring hospitalisation. Both SAEs resolved without sequelae and were deemed by the investigator to be unrelated to study drug administration.

Table 3-3. DARE-C II: Safety parameters

Total treated population Safety parameters (n=19)

Clinical parameters Total number of adverse events 41 Grade 3 or 4, n (%) 1 (2) Serious adverse events, n (%) 2 (11) Discontinuation due to adverse event, n (%) 0

Clinical adverse events Common (>10% of study population), n (%) Headache 4 (21) Fatigue 3 (16) Insomnia 3 (16) Nausea 3 (16) Sleep disorder 3 (16) Diarrhoea 2 (11) Musculoskeletal chest pain 2 (11) Pruritus 2 (11) Rash 2 (11)

Laboratory parameters Median reduction in Hb at end of treatment, g/L (IQR) 14 (2-23)

Decrease in Hb >30g/L, n (%) a 2 (11)

Decrease in Hb <100g/L, n (%) 2 (11) a Decrease in Hb >30g/L between baseline and end of treatment Abbreviations: Hb, haemoglobin

Page 120 of 309 Longitudinal behavioural assessment Injecting drug use risk behaviour (in the past 30 days) was assessed during study follow up. The proportion reporting recent injecting drug use was 32% (n=6) prior to enrolment, 42% (n=8) prior to baseline, 32% (n=6) prior to end of treatment, and 32% (n=6) prior to post- treatment week 12. The proportion reporting needle and syringe borrowing was 33% (n=2) prior to enrolment, 29% (n=2) prior to baseline and 20% (n=1) prior to end of treatment. No- one reported needle and syringe borrowing between end of treatment and post-treatment week 12. There was no significant change in injecting drug use (p=0.317) or needle and syringe borrowing (p=0.157) between enrolment and post-treatment week 12.

Page 121 of 309 Discussion While six weeks of sofosbuvir and ribavirin was safe and well tolerated, efficacy was poor with SVR12 only 32%. This study conclusively demonstrates that while on-treatment virological suppression was achieved in the majority, a high proportion of participants demonstrated post- treatment relapse. Baseline HCV RNA ≤6 log10 IU/mL and subsequent rapid viral suppression (HCV RNA below the LLoQ [TND or TDnq] at week 1 and HCV RNA TND at week 2) were associated with SVR12, which supports further research with more potent DAA regimens in recent HCV infection. In this cohort, while those participants achieving SVR12 did have significantly lower HCV RNA at baseline, spontaneous clearance was unlikely, given the median duration of HCV infection of 37 weeks.

Limited evidence exists for the use of DAA therapy in the setting of acute HCV infection with no published interferon-free DAA trials to date. Fierer et al performed a pilot study in HIV- positive MSM with recent HCV genotype 1 infection and administered PEG-IFN, ribavirin and telaprevir (308). Risk factors for acquisition were not reported. By ITT, SVR12 was 84% (16/19). Treatment duration was response-guided; of those who achieved SVR12, most (13/16; 81%) received 12 weeks of therapy. In the DARE-C I open-label pilot study, response-guided therapy (8-24 weeks) with PEG-interferon, ribavirin and telaprevir for recent HCV infection (duration of infection 6-18 months) was effective in the majority (SVR12 71%; 10/14), regardless of HIV co-infection, although similar to that observed with PEG-IFN and ribavirin alone in this population (332).

Interferon-free DAA therapy offers significant promise for individuals with recent HCV infection. DARE-C II is one of the first studies of interferon-free DAA therapy in this population although the regimen used in this study (a single DAA combined with ribavirin) is now regarded as suboptimal in patients with chronic HCV genotype 1 and genotype 3 infection (333). In contrast, combinations of 2 or more different classes of DAAs have achieved SVR in greater than 95% of individuals with chronic HCV infection (228, 230, 239). Preliminary results from recent pilot studies have reported encouraging results with short duration DAA therapy in acute HCV infection. In symptomatic acute HCV genotype 1 mono-infection, very high SVR was demonstrated with 6 weeks of sofosbuvir/ledipasvir (SVR12 ITT 100%, 20/20);

PWID were excluded (334). Notably, baseline HCV RNA was low in the majority (≤6 log10

IU/mL, 90%; ≤3 log10 IU/mL, 45%) and the proportion who may have demonstrated spontaneous clearance is unclear. Again, in acute HCV genotype 1 mono-infection (PWID included), high SVR was demonstrated with 4 weeks of sofosbuvir/ledipasvir (SVR12 ITT 100%, 14/14) and 8 weeks of sofosbuvir plus simeprevir (SVR12 ITT 87%, 13/15) (335). In keeping with our findings, viral suppression was rapid with HCV RNA below the limit of

Page 122 of 309 detection in 93% at week 1 in both arms. Similarly, in acute HCV genotype 1 and genotype 4 with HIV co-infection, SVR12 ITT was 77% (20/26; relapse, n=3; reinfection, n=1; LTFU, n=2) following 6 weeks of sofosbuvir/ledipasvir (SVR12 per-protocol analysis: 83% [20/24]) (336). Consistent with our results, the three participants with relapse had high baseline HCV

RNA (>6.9 log10 IU/mL), suggesting that even with potent DAA regimens, viral suppression may be protracted in those with a high HCV burden. Again, these data support further research with contemporary DAA regimens in acute HCV infection.

Internationally, increasing HCV incidence has been observed in HIV-positive MSM, largely associated with sexual and non-injecting drug use behaviour (11, 12). High reinfection incidence following SVR has been observed in some cohorts (269, 270). Prior to post-treatment week 12, one case of reinfection was demonstrated in an HIV-positive male, who reported concomitant injecting drug use (with sharing of needles, syringes and other paraphernalia) and high-risk sexual behaviours (including multiple casual male partners, group sex and unprotected anal intercourse). The risk for reinfection following treatment for recent HCV infection in those individuals with ongoing high risk behaviour emphasises the need for post-treatment surveillance, harm reduction strategies and education (270, 311). Of note, no change in injecting drug use behaviour was observed in participants longitudinally in this study; follow up is ongoing. Despite concerns regarding non-adherence and reinfection following treatment in PWID, the available evidence would indicate that active drug use does not significantly compromise the long term benefit of treating recent HCV infection with either interferon- containing or interferon-free regimens (246, 251, 312). Individuals with recent HCV infection should be offered antiviral therapy together with education and harm minimisation, in order to reduce high-risk behaviours (299) and the subsequent risk of reinfection.

The potential for broad access to highly effective, well tolerated interferon-free DAA regimens has stimulated discussion around HCV treatment-as-prevention and subsequent HCV elimination. Modelling suggests that substantial reductions in incidence and chronic HCV prevalence could be achieved by targeted DAA treatment scale-up amongst PWID and HIV- positive MSM at high risk of ongoing transmission (75, 232, 291). Importantly (and in opposition to many country and region specific DAA restrictions), a treatment scale-up model in HIV-positive MSM predicted that the greatest impact on HCV incidence and prevalence would be achieved if treatment was prioritised to those with recently diagnosed (<1 year) HCV infection, regardless of disease stage, and occurred when combined with behavioural interventions (75). Further, modelling has demonstrated the cost-effectiveness of DAA treatment scale-up among active PWID (232). International guidelines recommend that “treatment should be prioritized regardless of the fibrosis stage for individuals at risk of

Page 123 of 309 transmitting HCV, including active injection drug users” (103). However, due to high drug pricing, access to interferon-free DAA therapy is restricted, even in high-income settings: by and within countries, by fibrosis stage and by former or current substance misuse (307). If HCV elimination is to be achieved, expedient, widespread access to DAA therapy, without liver disease stage or drug use restriction, will be required.

Sample size and the applicability of the DAA regimen used may be considered study limitations. Given the pace of change in HCV therapeutics, the DAA regimen used, sofosbuvir plus ribavirin, is suboptimal in chronic HCV genotype 1 and genotype 3 infection (333). However, DARE-C II was designed as a proof-of-concept study of interferon-free DAA therapy in individuals with recent HCV infection. It confirmed the feasibility of ultrashort interferon- free DAA therapy in those with recent HCV infection, regardless of treatment regimen, with excellent adherence in this historically difficult-to-treat population. DARE-C II has informed future trial design in this population, exemplified by an upcoming international randomised control trial of ultrashort versus standard duration sofosbuvir/velpatasvir for recent HCV infection (NCT02625909). Despite the small sample size, the study also conclusively demonstrated the suboptimal efficacy of sofosbuvir and ribavirin for only six weeks.

The treatment paradigm for individuals with HCV infection is evolving rapidly. Combining two or more potent DAAs from different classes has increased SVR (>95%) and shortened treatment duration to only 8 weeks in most populations with chronic HCV infection (228, 230, 239). While these excellent results in chronic HCV infection have cast some doubt on any “efficacy advantage” of early treatment in acute HCV infection (140), the potential reduction in transmission amongst PWID and HIV-positive MSM will be beneficial (75, 300). HCV treatment-as-prevention strategies will be enhanced by early diagnosis and increased treatment uptake in recent HCV infection.

Page 124 of 309 Supplementary Material

Methods Study participants and setting Participants were enrolled between October 2014 and May 2015 through a network of tertiary hospitals in Australia (n=4) and New Zealand (n=1).

Adults (age ≥18 years) with recent HCV infection and HCV RNA ≥10,000 IU/mL at screening were eligible for study inclusion. Individuals with HIV co-infection were eligible provided: 1. HIV infection >6 months duration and 2. CD4 count >200 cells/mm3 and HIV viral load <50 copies/ml on stable combination antiretroviral therapy (cART) or 3. CD4 count ≥500 cells/mm3 and HIV viral load <100,000 copies/mL not on cART. The following antiretroviral agents were not permitted based on drug-drug interactions and potential adverse events: didanosine (327), zidovudine (328) and ritonavir-boosted tipranavir (210). Individuals with acute or chronic HBV co-infection were excluded.

Other exclusion criteria included: pregnant women or male partners of pregnant women; breast feeding; systemic anti-neoplastic or immunomodulatory therapy ≤6 months prior to first dose of study drug; any investigational drug ≤6 weeks prior to first dose of study drug; positive anti- HAV IgM Ab or anti-HBc IgM antibody at screening; alternative aetiology of chronic liver disease; decompensated liver disease; hepatocellular carcinoma (HCC); prior treatment with HCV protease or polymerase inhibitors; severe retinopathy; severe seizure disorder; immunologically mediated disease, chronic pulmonary disease with functional limitation, severe cardiac disease, organ transplantation (apart from corneal, skin or hair graft), malignancy, or other severe illness (including psychiatric) which in the opinion of the investigator would compromise the participants safety or ability to comply with the protocol; and the following lab values at screening: neutrophil count <1500 cells/mm3, platelet count <90,000 cells/mm3, creatinine >1.5 times the upper limit of normal (ULN), haemoglobin <12 g/dL in women or <13 g/dL in men, haemoglobin A1c ≥8.5%, International Normalised Ratio (INR) ≥1.5, serum albumin <33 g/L, serum total bilirubin >1.8 times the ULN (unless isolated in subjects with Gilbert’s syndrome).

Study definitions HCV virological suppression was defined as HCV RNA below the lower limit of quantitation (LLoQ) (target not detected [TND] or target detected, not quantifiable [TDnq]). An end-of- treatment response (ETR) was defined as serum HCV RNA below the LLoQ at the end of treatment. HCV virological failure was defined as non-response (failure of virological

Page 125 of 309 suppression on-treatment with quantifiable HCV RNA at all time points between baseline and end of treatment), breakthrough (an increase from non-quantifiable to quantifiable HCV RNA or to at least 1 log10 above nadir while on treatment) or post-treatment relapse (the presence of quantifiable HCV RNA after an ETR, confirmed as homologous virus on sequencing of Core- E2 and/or NS5B regions as described previously) (302, 303). Reinfection was defined by the presence of quantifiable HCV RNA after an ETR and detection of infection with an HCV strain that was distinct from the primary infecting strain (heterologous virus on sequencing of Core-E2 and/or NS5B regions). Loss of HIV virological control was defined as a confirmed HIV RNA of at least 400 copies/mL in individuals on cART.

The presentation of recent HCV infection at the time of diagnosis was classified as either acute clinical or asymptomatic infection. Acute clinical infection included participants with a documented clinical history of symptomatic seroconversion illness (including, but not limited to, the presence of jaundice, nausea/vomiting, abdominal pain, fever and hepatomegaly) and those without clinical symptoms but with a documented peak ALT >400 U/L within the 12 months prior to diagnosis. Asymptomatic infection included participants with anti-HCV antibody seroconversion but no acute clinical symptoms or documented peak ALT >400 U/L.

The duration of HCV infection at screening and baseline was calculated from the estimated date of infection. The estimated date of clinical infection was calculated as six weeks before onset of seroconversion illness or six weeks before the first ALT >400 U/L. The estimated date of asymptomatic infection was calculated as the midpoint between the last negative anti-HCV antibody and the first positive anti-HCV antibody. For participants who were anti-HCV antibody negative and HCV-RNA positive at screening, the estimated date of infection was six weeks before enrolment, regardless of symptom status.

On-treatment adherence was calculated for each medication individually (sofosbuvir and ribavirin) by subtracting the number of missed doses from the total number of doses prescribed for therapy duration and dividing by the total number of doses prescribed for therapy duration. By pill count and self-reported questionnaire, compliance with sofosbuvir and ribavirin were individually calculated at the 90/90 and 100/100 adherence levels, defined as receipt of ≥90 or 100% of scheduled sofosbuvir or ribavirin doses for ≥90 or 100% of the scheduled treatment period, respectively.

Page 126 of 309 Tables

Supplementary Table 3-1. DARE-C II: Clinical and virological characteristics of excluded participants

Screening Screen HCV Gender Age HIV HCV RNA Reason for exclusion date GT (IU/ml)

Ineligible (duration of 23/10/2014 Male - No 3a 64 919 infection >12 months)

No sample 25/11/2014 Male 48 Yes 2a Poor venous access taken

18/12/2014 Male 43 Yes 1a 4 600* HCV RNA <10,000

19/12/2014 Male 36 Yes 1a TND HCV RNA TND

12/02/2015 Female 25 No 3a TND HCV RNA TND

Psychiatric admission 26/02/2015 Female 37 No 1a 3 240 000 after screening

7/05/2015 Female 18 No 3a 177 000 Withdrawal of consent

*HCV RNA <15 IU/mL (target detected, not quantifiable) on re-screening 4 weeks later

Abbreviations: GT, genotype; TND, target not detected.

Page 127 of 309 Supplementary Table 3-2. DARE-C II: Primary and secondary efficacy endpoints, by HIV co- infection

Total treated HCV HIV/HCV Efficacy population mono-infection co-infection (n=19) (n=5) (n=14) ETR, n (%) 17 (89) 5 (100) 12 (86) SVR4, n (%) 8 (42) 3 (60) 5 (36)

SVR12, n (%) 6 (32) 3 (60) 3 (21) Non-response 2 (11) 0 2 (14) Relapse 9 (47) 2 (40) 7 (50) Reinfection 1 (5) 0 1 (7) Lost to follow up 1 (5) 0 1 (7)

SVR24, n (%) 7 (37)* 3 (60) 4 (29)*

Viral kinetics on treatment, n (%) HCV RNA

*Note, confirmed spontaneous clearance of HCV viraemia between post-treatment week 12 and 24 in one HIV-positive participant

Supplementary Table 3-3. DARE-C II: Primary and secondary efficacy endpoints, by HCV genotype

Genotype 1 Genotype 2 Genotype 3 Efficacy (n=13) (n=1) (n=5) ETR, n (%) 12 (92) 1 (100) 4 (80) SVR4, n (%) 4 (31) 1 (100) 3 (60)

SVR12, n (%) 3 (23) 1 (100) 2 (40) Non-response 1 (8) 0 1 (20) Relapse 8 (62) 0 1 (2) Reinfection 0 0 1 (20) Lost to follow up 1 (8) 0 0

SVR24, n (%) 3 (23) 1 (100) 3 (60)*

Viral kinetics on treatment, n (%) HCV RNA

*Note, confirmed spontaneous clearance of HCV viraemia between post-treatment week 12 and 24 in one HIV-positive participant

Page 128 of 309 Supplementary Table 3-4. DARE-C II: Changes in key laboratory parameters between screening and post treatment week 12

Post Laboratory parameter, median End of Screening Baseline treatment (range) treatment week 12 139 133 22 48 ALT, U/L (60 – 1040) (25 – 425) (8 – 44) (13 – 910) 88 71 27 33 AST, U/L (31-478) (27-320) (14-39) (21-290) 9 11 11 11 Bilirubin (total), umol/L (5-39) (4-49) (6-44) (4-50) 153 146 134 149 Haemoglobin, g/L (131-170) (126-171) (90-160) (133-173) 3.4 2.9 3.4 3.0 Absolute neutrophil count, x109/L (2.0-7.5) (1.7-7.4) (1.8-8.0) (1.7-9.4) 226 231 261 230 Platelet count, x109/L (149-423) (145-349) (158-404) (137-333) 598 614 728 CD4 count*, x106/L - (370-986) (283-838) (415-1340) *HIV-positive participants only

Normal range for laboratory values: ALT, 0-30 U/L; AST, 0-30 U/L; absolute neutrophil count, 2.0 - 7.5 x109/L; Bilirubin, 0-18 umol/L; CD4 count, 500 - 1650 x106/L; Haemoglobin, female: 115 - 165 g/L, male: 130 - 180 g/L; Platelet count, 150-400 x109/L.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase

Page 129 of 309 Figures

A 8

6 (IU /m L) 10

4 HCV RNAHCV log

2

LLoQ LLoD

0 SCR BL D2 W1W2 W4 EOT PT W4 PT W12 PT W24

B 8

6 (IU /m L) 10

4 HCV RNAHCV log

2

LLoQ LLoD

0 SCR BL D2 W1W2 W4 EOT PT W4 PT W12 PT W24

Page 130 of 309 C 8

6 (IU /m L) 10

4 HCV RNAHCV log

2

LLoQ LLoD

0 SCR BL D2 W1W2 W4 EOT PT W4 PT W12 PT W24

D 8

6 (IU /m L) 10

4 HCV RNAHCV log

2

LLoQ LLoD

0 SCR BL D2 W1W2 W4 EOT PT W4 PT W12 PT W24

Supplementary Figure 3-1. DARE-C II: On-treatment viral kinetics

Panel A: Sustained virological response (n=6). Panel B: Relapse (n=9). Panel C: Non-response (n=2). Panel D: Reinfection (n=1; depicted by open circles) and LTFU (n=1). Grey shaded area indicates period on treatment.

Abbreviations: BL, baseline; D2, day 2; EOT, end of treatment; LLoD, lower limit of detection); LLoQ, lower limit of quantitation; PT, post treatment; SCR, screening; SVR, sustained virological response; W1- 4, week 1-4

Page 131 of 309 Supplementary Figure 3-2. DARE-C 1200 II: Change in serum alanine 1000 aminotransferase (ALT), aspartate 800 600 aminotransferase (AST) and total bilirubin prior to, on and post-treatment

500 Panel A: ALT. 400 Panel B: AST. Panel C: Total bilirubin.

ALT (U/L) 300 Bars depict median with interquartile range. Dotted line at upper limit of normal (ULN) 200 for each parameter - ALT and AST, ULN 30 U/L; total bilirubin ULN 18 umol/L. In 100 Panel C, black circle indicates the single HIV-positive participant receiving ritonavir- 0 boosted atazanavir. SCR BL W1 W2 W4 EOT PT W 12 At end of treatment, two participants had ALT above the ULN. The first participant

600 (ALT 35 U/L) achieved SVR12 with ALT within normal limits at post treatment week 12 (26 U/L). The second participant (ALT 44 U/L) demonstrated post treatment relapse with ALT rising to 153 U/L at post treatment week 12. 400

Abbreviations: Alanine aminotransferase (ALT); aspartate aminotransferase (AST); AST (U/L) baseline (BL); end of treatment (EOT); post

200 treatment week 12 (PT W12); screening (SCR); upper limit of normal (ULN)

0 SCR BL W1 W2 W4 EOT PT W 12

80

60

40 B ilirubin (um ol/L )

20

0 SCR BL W1 W2 W4 EOT PT W 12

Page 132 of 309 8

SVR12 (n=6)

Relapse (n=9)

6

4 HCV RNA, IU/mL RNA, HCV 10

2 Mean log LLoQ LLoD

0 1 2 7 14 21 28 42 Tim e, days

Supplementary Figure 3-3. DARE-C II: Geometric mean decline in HCV RNA on treatment

Page 133 of 309

Treatment Treatment period (weeks) SVR12 duration Sofosbuvir Ribavirin (weeks) 0-2 2-4 4-6 0-2 2-4 4-6

2 No 6 No 6 No 6 No 6 * No 6 * * Yes 6 No 6 * No 6 * * Yes 6 * Yes 6 * * Yes 6 * No 6 * No 6 * No 6 * * Yes 6 * No 6 * * Yes 6 * No 6 * No

SVR12 indicated by * Figure 90-99% 80-90% 80-50% 100% dose 1-50% dose No dose legend dose dose dose

Supplementary Figure 3-4. DARE-C II: On-treatment adherence

Abbreviations: SVR, sustained virological response

Page 134 of 309 A

6

4

2

Ribavirin plasma concentration (mg/L) concentration plasma Ribavirin 0 Hour 4 Day 2 W eek 1 W eek 2 W eek 4 EOT

B

6

4

2

Ribavirin plasma concentration (mg/L) concentration plasma Ribavirin 0 Hour 4 Day 2 W eek 1 W eek 2 W eek 4 EOT

Supplementary Figure 3-5. DARE-C II: Ribavirin plasma concentration (mg/L) by treatment time-point and virological outcome

Panel A: Participants achieving SVR12 (n=6). Panel B: Participants with virological failure (n=13). Bars depict median with interquartile range. Dotted line at ribavirin plasma concentration 2mg/L.

Page 135 of 309 Chapter 4 HCV reinfection incidence among individuals treated for recent infection

Chapter Introduction and Objectives Highly effective, well tolerated interferon-free direct-acting antivirals have revolutionised HCV therapeutics. Modelling suggests that significant reductions in HCV incidence and prevalence could be achieved by targeted treatment scale-up among those at high risk of transmission. One challenge to HCV elimination though therapeutic intervention is reinfection.

The incidence of and factors associated with HCV reinfection following treatment for recent infection were assessed in individuals who achieved an end-of-treatment response in four open- label studies between 2004 and 2015 in Australia and New Zealand (ATAHC I, ATAHC I, DARE-C I, DARE-C II). In the cohort at-risk for reinfection, ten cases of reinfection were identified, for an incidence of 7.4 per 100 py (95% CI 4.0, 13.8). Reinfection incidence was significantly higher amongst those who reported injection drug use at end of or post-treatment.

High reinfection incidence following treatment for recent HCV infection in individuals with ongoing risk behaviour emphasises the need for post-treatment surveillance, harm reduction strategies and education in at-risk populations. Further, access to antiviral therapy for treatment of HCV reinfection will be crucial in the context of HCV elimination strategies.

Publication Marianne Martinello, Jason Grebely, Kathy Petoumenos, Edward Gane, Margaret Hellard, David Shaw, Joe Sasadeusz, Tanya L Applegate, Gregory J Dore, Gail V Matthews. HCV reinfection incidence among individuals treated for recent infection. J Viral Hepatitis. 2016. [Accepted article].

Page 136 of 309 Declaration I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Marianne Martinello

Viral Hepatitis Clinical Research Program Kirby Institute, UNSW Australia Wallace Wurth Building, Sydney NSW 2052 t: +61 413 276 968 e: [email protected]

Page 137 of 309 Co-authorship Acknowledgement In the case of Chapter Four, the nature and extent of my contribution to the work was the following:

Author name Contribution (%) Nature of contribution

Conducted the data analysis, contributed to study design, data collection and led the Marianne Martinello 50 development, writing and critical revision of the manuscript

Contributed to study design, interpretation of Jason Grebely 3 findings and critical revision of the manuscript

Kathy Petoumenos 2 Contributed to statistical analysis of data

Contributed to study design, implementation, Edward Gane 2 data collection and critical revision of the manuscript

Contributed to study design, implementation, Margaret Hellard 2 data collection and critical revision of the manuscript

Contributed to study design, implementation, David Shaw 2 data collection and critical revision of the manuscript

Contributed to study design, implementation, Joe Sasadeusz 2 data collection and critical revision of the manuscript

Contributed to study design and critical revision Tanya Applegate 2 of the manuscript

Contributed to study design, data collection, Gregory J Dore 15 interpretation of findings and critical revision of the manuscript

Contributed to study design, data collection, Gail V Matthews 15 interpretation of findings and critical revision of the manuscript

Page 138 of 309 HCV reinfection incidence among individuals treated for recent infection

Marianne Martinello1, Jason Grebely1, Kathy Petoumenos1, Edward Gane2, Margaret Hellard3,4,5, David Shaw6, Joe Sasadeusz7, Tanya L Applegate1, Gregory J Dore1,8, Gail V Matthews1,8

1. Viral Hepatitis Clinical Research Program, Kirby Institute, UNSW Australia, Sydney, NSW, Australia 2. Auckland Hospital, Auckland, New Zealand 3. Centre for Population Health, Burnet Institute, Melbourne, VIC, Australia 4. Infectious Diseases Unit, Alfred Hospital, Melbourne, VIC, Australia 5. Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, VIC, Australia 6. Infectious Diseases Unit, Royal Adelaide Hospital, Adelaide, SA, Australia 7. Victorian Infectious Diseases Service, Royal Melbourne Hospital, Melbourne, VIC, Australia 8. Department of Infectious Disease and Immunology, St Vincent’s Hospital, Sydney, NSW, Australia

Page 139 of 309 Abstract Background and Objectives: One challenge to HCV elimination through therapeutic intervention is reinfection. The aim of this analysis was to calculate the incidence of HCV reinfection among both HIV-positive and negative individuals treated for recent HCV infection (estimated infection duration <18 months).

Methods: Individuals with recent HCV infection who achieved an end-of-treatment response in four open-label studies between 2004 and 2015 in Australia and New Zealand were assessed for HCV reinfection, confirmed by sequencing of the Core-E2 and/or NS5B regions. Reinfection incidence was calculated using person-time of observation. Exact Poisson regression analysis was used to assess factors associated with HCV reinfection.

Results: The cohort at-risk for reinfection (n=120; 83% male; median age 36 years) was composed of HIV-positive men-who-have-sex-with-men (53%) and people who inject drugs (current 49%, ever 69%). Total follow-up time at-risk was 135 person-years (median 1.08 years, range 0.17, 2.53). Ten cases of HCV reinfection were identified, for an incidence of 7.4 per 100 py (95% CI 4.0, 13.8). Reinfection incidence was significantly higher amongst participants who reported injection drug use at end of or post-treatment, irrespective of HIV status (15.5 per 100 py, 95% CI 7.8, 31.1). In adjusted analysis, factors associated with reinfection were older age (aIRR 5.3, 95% CI 1.15, 51.5, p=0.042) and injection drug use at end of or post-treatment (aIRR 7.9, 95%CI 1.6, 77.2, p=0.008).

Conclusions: High reinfection incidence following treatment for recent HCV infection in individuals with ongoing risk behaviour emphasises the need for post-treatment surveillance, harm reduction strategies and education in at-risk populations.

Registration: ClinicalTrials.gov registry ATAHC I: NCT00192569; ATAHC II: NCT01336010; DARE-C I: NCT01743521; DARE-C II: NCT02156570

Page 140 of 309 Introduction Highly effective, well tolerated interferon-free direct-acting antivirals (DAA) have revolutionised hepatitis C virus (HCV) therapeutics (140), with daily fixed-dose combination DAA regimens providing cure in greater than 95% of individuals with chronic infection (228, 229). The availability of DAA therapy has led to significant therapeutic optimism with the possibility of broad treatment uptake and subsequent HCV elimination (75, 231-233). One challenge to HCV elimination though therapeutic intervention is reinfection.

There is concern that HCV reinfection may compromise the individual and population level benefits of HCV treatment in some populations with the risk of reinfection cited as a reason for not offering treatment to people who inject drugs (PWID) (266, 267). However, in general, the incidence of HCV reinfection in PWID treated for chronic HCV infection ranges between one and five per 100 py (summarised in Supplementary Table 4-1). Reinfection incidence following treatment in individuals with HIV/HCV co-infection is varied, with high incidence reported in some cohorts of HIV-positive men-who-have-sex-with-men (MSM) (12, 269, 270). There is uncertainty around these reinfection estimates due to sample size, retrospective study designs, exclusion of recent PWID from trials, varied definitions for recent injection drug use and time at-risk for reinfection, and the inability to accurately distinguish relapse from reinfection.

Mathematical modelling suggests that substantial reductions in HCV incidence and prevalence could be achieved by targeted DAA treatment scale-up amongst those at highest risk of ongoing transmission, including PWID and HIV-positive MSM with recently diagnosed HCV infection (75, 271, 291, 292). Despite the high cost of DAA therapy, treating recent PWID and HIV- positive MSM with early liver disease appears to be cost-effective compared to delaying until cirrhosis, given the reduction in liver-related complications and additional benefit of averting secondary infections (232, 233, 293). However, ongoing risk behaviours associated with HCV transmission may contribute to reinfection and compromise the population-level benefits of Treatment as Prevention (75, 294, 297). Few studies have evaluated the incidence of HCV reinfection following treatment of recent HCV infection (summarised in Supplementary Table 4-1) (269, 270, 285), a high-risk group for onward transmission and of importance as DAA treatment access expands to traditionally marginalised populations. The aim of this analysis was to calculate the incidence of HCV reinfection among individuals treated for recent HCV infection (estimated infection duration <18 months) and assess clinical and behavioural factors associated with reinfection.

Page 141 of 309 Methods Study participants Individuals with recent HCV infection (infection duration <18 months) who received treatment in four prospective open-label studies (ATAHC I, ATAHC II, DARE-C I and DARE-C II) between 2004 and 2015 in Australia and New Zealand were assessed for HCV reinfection (133, 332, 337) (Figure 4-1). The primary endpoints of these studies (133, 332, 337) and an analysis of HCV superinfection and reinfection in treated and untreated participants in ATAHC I (285) have been published previously.

Recent primary HCV infection, n=278

ATAHC I (untreated), n=52 ATAHC II (untreated), n=30

DARE-C I, n=14 ATAHC I (treated), ATAHC II (treated), DARE-C II, n=19 n=111 n=52 PEG-IFN, RBV + telaprevir Sofosbuvir + RBV PEG-IFN +/- RBV PEG-IFN + RBV (response guided; 8 - (6 weeks) (24 weeks) (response guided; 8 - 24 weeks) Duration of HCV Duration of HCV 48 weeks) Duration of HCV infection ≤12 infection ≤18 Duration of HCV infection 6 - 18 months months infection ≤18 months months

End of treatment End of treatment End of treatment End of treatment response, n=79 response, n=43 response, n=12 response, n=17

SVR, n=61 SVR, n=34 SVR, n=9 SVR, n=6

Reinfection, n=5 Reinfection, n=3 Reinfection, n=1 Reinfection, n=1

Relapse, n=8 Relapse, n=6 Relapse, n=2 Relapse, n=9

LTFU, n=5 LTFU, n=1

Figure 4-1. Participant disposition

Participants highlighted in bold constitute the cohort at-risk for reinfection.

Page 142 of 309 Recent primary HCV infection was defined as initial detection of serum anti-HCV antibody and/or HCV RNA within six months of enrolment and either (i) documented recent HCV seroconversion (anti-HCV antibody negative result in the 18 [DARE-C II] or 24 [ATAHC, ATAHC II, DARE-C I] months prior to enrolment) or (ii) acute clinical hepatitis (jaundice or alanine aminotransferase [ALT] greater than 10 times the upper limit of normal [ULN]) within the previous 12 months with the exclusion of other causes of acute hepatitis, with estimated duration of HCV infection less than 12 [DARE-C II] or 18 [ATAHC, ATAHC II, DARE-C I] months at screening.

HCV RNA testing and sequencing The presence of HCV RNA was assessed at all scheduled study (see Supplementary Material). In ATAHC I, HCV RNA was assessed using a qualitative HCV RNA assay (Versant transcription-mediated amplification [TMA]; Bayer, Australia; LLoD 10 IU/ml) and if positive, a quantitative HCV RNA assay (Versant HCV RNA 3.0; Bayer, Australia; LLoD 615 IU/ml). In ATAHC II, DARE-C I and DARE-C II, HCV RNA was assessed using a quantitative HCV RNA assay (COBAS Taqman 2.0; Roche Diagnostics, USA; LLoD 15 IU/mL). Population- based (Sanger) HCV RNA sequencing was performed on the first available pre-treatment quantifiable HCV RNA sample and the first available quantifiable HCV RNA sample following HCV RNA recurrence using an in-house assay with methods described previously (302, 303). See Supplementary Material for more details.

Study definitions and outcomes An end-of-treatment response (ETR) was defined as HCV RNA below the lower limit of detection, target not detected (LLoD, TND). HCV RNA recurrence was defined as quantifiable HCV RNA following ETR. Post-treatment relapse was defined as the presence of quantifiable HCV RNA after an ETR, confirmed as homologous virus on sequencing of Core-E2 and/or NS5B regions as described (302, 303). Confirmed reinfection was defined by the presence of quantifiable HCV RNA after an ETR with detection of an HCV strain that was distinct from the primary infecting strain (heterologous virus on sequencing). Possible reinfection was defined by the presence of quantifiable HCV RNA after an ETR without sequence data, but occurring after post treatment week 24 and with documentation of HCV RNA TND at post treatment week 12 or 24 to exclude post-treatment relapse. Persistent reinfection was defined by the presence of quantifiable HCV RNA in a repeated sample taken at least 12 weeks after HCV RNA recurrence.

The time at risk for reinfection was calculated from the date of end of treatment in individuals with an ETR to date of reinfection or last undetectable HCV RNA. The estimated date of

Page 143 of 309 reinfection was calculated as the midpoint between the dates of the last undetectable HCV RNA test and the first quantifiable HCV RNA test during follow-up. The primary study outcome was HCV reinfection incidence.

Study oversight All study participants provided written informed consent before study procedures. The study protocols were approved by St Vincent’s Hospital, Sydney Human Research Ethics Committee (primary study committee), as well as local ethics committees at all study sites. The studies were conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines. The studies were registered with clinicaltrials.gov registry (ATAHC I: NCT00192569; ATAHC II: NCT01336010; DARE-C I: NCT01743521; DARE-C II: NCT02156570).

Statistical analysis In the cohort at-risk for reinfection (ETR without post-treatment relapse), categorical parameters were summarised as number and proportion and continuous variables were summarised by either mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. Reinfection incidence was calculated using person-time of observation. Confidence intervals (CI) for rates were calculated using Poisson distribution.

Exact Poisson regression analysis was used to assess factors associated with HCV reinfection, with time at risk (years) as the exposure variable. In unadjusted analyses, potential predictors were determined a priori and included sex, age at study enrolment, income, education level, social functioning score at enrolment (median), mode of HCV acquisition (injection drug use, sexual, other), HIV infection, injection drug use (ever, previous 6 months at enrolment, previous 30 days at enrolment), and injection drug use at end of and/or post-treatment. Social functioning was calculated using a validated scale from the Opiate Treatment Index (313) and addressed employment, residential stability, interpersonal conflict and social support (higher score reflects poorer social functioning, range score 0-48). All variables with p<0.2 in univariate analysis were considered in multivariate regression models using a backwards stepwise approach. Statistically significant differences were assessed at p<0.05; p-values were two-sided. Additional models assessed factors associated with reinfection among lifetime PWID (participants who reported injection drug use at least once) and HIV-positive MSM. See Supplementary Material for more details.

Analysis was performed using STATA version 14.0 (StataCorp, College Station, TX).

Page 144 of 309 Results Participant disposition Between 2004 and 2015, 278 participants with recent HCV infection were enrolled in Australia and New Zealand with 196 participants included in the intention-to-treat population; 82 participants were enrolled into the untreated arms of ATAHC I and II (Figure 4-1). An end of treatment response (ETR) was documented in 77% (n=151). Six participants (4%) were lost to follow up (LTFU) after ETR. Viral recurrence following ETR was seen in 35 (23%), confirmed by sequencing as relapse in 25 (17%) and reinfection in 10 (6%). Participants with relapse and LTFU after ETR were excluded from subsequent analysis.

The enrolment characteristics of the cohort at-risk for reinfection (n=120; male 83%; median age 36 years, IQR 29-46) are shown in Table 4-1. HIV co-infection was documented in 53%; all of whom identified as MSM. Injection drug use ever prior to enrolment, within six months and within 30 days of enrolment was reported by 69%, 49% and 43%, respectively. Of those participants who reported injection drug use within 30 days of enrolment (n=51), the drugs most commonly injected were amphetamines (61%) and heroin or other opiates (29%) (Supplementary Table 4-2). Injection drug use at end of or post treatment was reported by 38% (n=45). Among those reporting injection drug use during follow-up, 71% (n=32) reported predominantly injecting amphetamines and 22% (n=10) reported use of unsterile needles and/or syringes. Different drug use behaviours were observed among participants with HIV/HCV co- infection as compared with HCV mono-infection; while participants with HIV/HCV co- infection were less likely to have ever injected drugs (61% vs 80%; p=0.021), those who did were older at commencement of injection drug use (30 years [IQR 25, 41] vs 23 years [IQR 18, 30]; p<0.001) (Supplementary Table 4-3).

Total follow-up time post treatment was 141 person-years (py; median 1.22 py, range 0.19, 2.53). Total follow-up time at-risk for reinfection (censured at estimated date of reinfection) was 135 py (median 1.08 years, range 0.17, 2.53).

Page 145 of 309 Table 4-1. Enrolment demographic and clinical characteristics of participants at risk for reinfection

HCV Demographic and clinical Overall No reinfectiona Reinfection characteristics N=120 N=110 N=10 Age at enrolment, median (IQR) 36 (29-46) 44 (36-49) 35 (24-46) Gender Male 100 (83) 10 (100) 90 (82) Female 19 (16) 0 19 (17) Transgender 1 (1) 0 1 (1) Full or part-time employment 68 (56) 5 (50) 63 (57) Tertiary education or greater, n (%) 70 (58) 8 (80) 62 (56) Social functioning score, median (IQR) 11 (6-16) 16 (12-17) 11 (6-15) HIV infection, n (%) 64 (53) 7 (70) 57 (52) On cART, n (%) 52 (81) 5 (71) 47 (82)

IDU, n (%) Ever prior to enrolment 83 (69) 7 (70) 77 (69) Previous 6 months prior to enrolment 59 (49) 6 (60) 53 (48) Previous 30 days prior to enrolment 51 (43) 6 (60) 45 (41) Age at first IDU, median (IQR) 25 (20-34) 35 (30-46) 25 (20-32) OST, n (%) Ever prior to enrolment 14 (12) 1 (10) 13 (13) At enrolment 6 (5) 1 (10) 5 (5)

Mode of primary HCV acquisition, n (%) IDU 66 (55) 6 (60) 60 (55) Sexual exposure 51 (43) 4 (40) 47 (43) Other 3 (3) 0 3 (3)

Weeks between estimated date of HCV infection and treatment commencement, 36 (30-46) 34 (27-52) 37 (30-46) median (IQR) HCV treatment PEG ± RBV 103 (86) 8 (80) 95 (86) PEG-IFN + RBV + telaprevir 10 (8) 1 (10) 9 (8) Sofosbuvir + RBV 7 (6) 1 (10) 6 (5) Treatment weeks, median (IQR) 24 (16-24) 20 (8-24) 24 (16-24)

Follow-up time post treatment (years), 1.22 1.44 1.19 median (IQR) (0.61, 1.52) 0.96, 1.97) (0.60, 1.52) Time at risk for reinfection (years), 1.08 0.67 1.19 median (IQR) (0.59, 1.50) (0.35, 1.23) (0.60, 1.52) a Includes participants with ETR and no reinfection or relapse

Abbreviations: IDU, injecting drug use; OST, opioid substitution therapy; PEG-IFN, pegylated interferon; RBV, ribavirin

Page 146 of 309 8

6 (IU/m L) 10

4 HCV RNAHCV log

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LLoQ LLoD

0 BL W2W4 ETRPT PT PT PT PT PT PT W4 W 12 W 24 W 48 W 72 W 96 W 120

6

4 (IU/m L) 10

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LLoQ LLoD

0 BL W2W4 ETRPT PT PT PT PT PT PT W4 W 12 W 24 W 48 W 72 W 96 W 120

8

6 (IU/m L) 10

4 HCV RNAHCV log

2

LLoQ LLoD

0 BL W2W4 ETRPT PT PT PT PT PT PT W4 W 12 W 24 W 48 W 72 W 96 W 120

Figure 4-2. Viral kinetics on and post-treatment in participants with HCV reinfection

Panel A: Persistent HCV reinfection. Panel B: Spontaneous clearance of HCV reinfection. Panel C: Indeterminate HCV reinfection outcome. Shaded area indicates period on treatment. LLoQ 25 IU/mL and LLoD 15 IU/mL for COBAS Taqman 2.0, Roche Diagnostics, USA. Abbreviations: BL, baseline; ETR, end-of-treatment response; LLoD, lower limit of detection; LLoQ, lower limit of quantitation; W, week; PT, post treatment

Page 147 of 309 HCV reinfection among participants treated for recent HCV infection Ten cases of HCV reinfection were identified (eight confirmed, two possible), with persistent reinfection in five and spontaneous clearance in three cases (Figure 4-2). Reinfection outcome was indeterminate in two cases due to lack of on-study follow-up testing. Of the ten participants (seven HIV-positive MSM) with reinfection, eight reported injection drug use during follow up; one HIV-positive MSM who reported never injecting drugs prior to study enrolment subsequently injected anabolic steroids during follow-up. The remaining two cases occurred in HIV-positive MSM who denied ever injecting drugs. Detailed demographic and clinical characteristics of the ten participants with reinfection are displayed in Table 4-2.

Among eight cases of confirmed reinfection, median estimated time to HCV reinfection was 35 weeks (range 9 - 81 weeks) from end of treatment. The shortest time to reinfection was noted in an HIV-positive MSM following short duration interferon-free DAA therapy who reported high-risk drug and sexual behaviour. Despite confirmation of sustained virological response at week 4 post treatment (SVR4, HCV RNA TND), HCV RNA was quantifiable at post treatment week 12 with an HCV genotype switch from 3a (screening) to 1a (post treatment week 12) in association with an acute clinical illness and transaminitis (ALT 910 U/L). Amongst participants with confirmed reinfection, median ALT at end of treatment and following diagnosis of reinfection were 25 U/L (range 15 – 83 U/L) and 192 U/L (range 15 – 1135 U/L), respectively.

Low level quantifiable HCV RNA was detected in the two cases of possible reinfection and as such, reinfection could not be confirmed by sequencing. Estimated time to possible reinfection was 31 and 64 weeks from end of treatment, respectively. In the first case, quantifiable HCV

RNA (3.87 log10 IU/mL) was detected at a single time point at post treatment week 36 (TND at post treatment week 24) and was repeatedly negative between post treatment weeks 48 and 120.

In the second case, quantifiable HCV RNA (2.18 log10 IU/mL) was detected at post treatment week 72 (TND at post treatment week 48) and was repeatedly negative subsequent to this.

Page 148 of 309 Table 4-2. Detailed demographic, behavioural and virological characteristics of participants with HCV reinfection

IDU HCV genotype Time ALT Mode of between Gender, Region Reinfection 1 HIV primary At EOT and age During sequenced outcome HCV screening Primary Reinfection reinfection EOT Reinfection 2 follow-up (weeks) Confirmed reinfection Male Spontaneous No IDU Yes Yes 3a 1a E1/HVR1 18 21 53 29 clearance Male Yes IDU Yes Yes 3a/1a 3a/1a Core-E2 81 83 1135 Persistence 49 Male E1/HVR1 No IDU Yes Yes 1a 3a 30 26 319 Persistence 36 Core-E2 Male Yes Sexual No No 2a 1a/3a Core-E2 40 58 565 Indeterminate 44 Male Yes Sexual No Yes 1a 1a NS5B 65 18 15 Indeterminate 50 Male Core-E2 Yes IDU Yes Yes 3a 1a 39 15 65 Persistence 62 NS5B Male Core-E2 Yes Sexual No No 1a 1a 18 29 54 Persistence 45 NS5B Male Core-E2 Yes IDU Yes Yes 3a 1a 9 23 910 Persistence 41 NS5B Possible reinfection Male Spontaneous No IDU Yes Yes 3a NA3 InnoLipa 31 15 14 35 clearance Male Spontaneous Yes Sexual No Yes4 1a NA3 InnoLipa 64 71 43 46 clearance 1 Age at study enrolment 3 Unable to sequence due to low HCV RNA at time of viral recurrence 2 IDU within 6 months of study enrolment 4 Anabolic steroids Abbreviations: ALT, alanine aminotransferase; EOT, end of treatment; IDU, injecting drug use1 ALT, normal range 0-30 U/L

Page 149 of 309 Among all participants at-risk for reinfection, reinfection incidence was 7.4 per 100 py (95% CI 4.0, 13.8) (Table 4-3 and Supplementary Table 4-4), for a projected cumulative reinfection incidence of 7.2% at one year and 14.5% at two years from end of treatment (Supplementary Figure 4-1). The incidence of HCV reinfection was 4.5 per 100 py (95% CI 1.4, 13.9) amongst those with HCV mono-infection, compared to 10.3 per 100 py (95% CI 4.9, 21.7) in those with HIV/HCV co-infection (p=0.232). The incidence of HCV reinfection was 8.5 per 100 py (95% CI 4.2, 16.9) in those who had ever injected drugs, compared to 4.9 per 100 py (95% CI 1.2, 19.8) in those who had never injected drugs (p=0.532). HCV reinfection incidence was significantly higher amongst participants who reported injecting drug use at end of and/or post treatment (15.5 per 100 py, 95% CI 7.8, 31.1) as compared with those who did not inject drugs during follow up (2.6 per 100 py, 95% CI 0.6, 10.3) (p=0.023).

Page 150 of 309 Table 4-3. Incidence of HCV reinfection among participants treated for recent HCV infection

Cases of Incidence Participants Participant type reinfection PYFU per 100 95% CI at risk (n) (n) py Overall

Confirmed/possible reinfection 10 120 135 7.4 4.0, 13.8 Confirmed reinfection 8 120 135 5.9 3.0, 11.9 Confirmed persistent reinfection 5 120 135 3.7 1.5, 8.9

HCV mono-infection

Confirmed/possible reinfection 3 56 67 4.5 1.4, 13.9 Confirmed reinfection 2 56 67 3.0 0.7, 11.9 Confirmed persistent reinfection 1 56 67 1.5 0.2, 10.6

HIV/HCV co-infection

Confirmed/possible reinfection 7 64 68 10.3 4.9, 21.7 Confirmed reinfection 6 64 68 8.9 4.0, 19.7 Confirmed persistent reinfection 4 64 68 5.9 2.2, 15.7

IDU ever

Confirmed/possible reinfection 8 84 94 8.5 4.2, 16.9 Confirmed reinfection 6 84 94 6.4 2.9, 14.1 Confirmed persistent reinfection 4 84 94 4.2 1.6, 11.3

IDU never

Confirmed/possible reinfection 2 36 41 4.9 1.2, 19.8 Confirmed reinfection 2 36 41 4.9 1.2, 19.8 Confirmed persistent reinfection 1 36 41 2.5 0.3, 17.5

IDU at end of and/or post-treatment

Confirmed/possible reinfection 8 45* 52 15.3 7.7, 30.6 Confirmed reinfection 6 45* 52 11.5 5.2, 25.5 Confirmed persistent reinfection 4 45* 52 7.7 2.9, 20.4

No IDU at end of and/or post-treatment

Confirmed/possible reinfection 2 72* 77 2.6 0.7, 10.4 Confirmed reinfection 2 72* 77 2.6 0.7, 10.4 Confirmed persistent reinfection 1 72* 77 1.3 0.2, 9.2 *Numbers do not equal 120 due to missing data on injecting during follow-up in 3 participants Abbreviations: CI, confidence interval; IDU, injecting drug use; py, person years; PYFU, total person years follow up

Page 151 of 309 Risk factors for HCV reinfection following treatment for recent infection Factors associated with HCV reinfection were assessed using exact Poisson regression analysis (Table 4-4). In adjusted analysis, factors independently associated with reinfection included older age (aIRR 5.42, 95% CI 1.06, 52.93, p=0.040) and injecting drug use at end of and/or post-treatment (aIRR 7.86, 95%CI 1.54, 76.79, p=0.008). Factors associated with reinfection were unchanged when the analysis was limited to those with confirmed reinfection (Supplementary Table 4-5).

Factors associated with HCV reinfection were also assessed amongst lifetime PWID (n=84; 46% HIV-positive MSM) and HIV-positive MSM (n=64). Among lifetime PWID, 54% reported injection drug use at end of and/or post-treatment. Median age at first injection drug use was 25 years (IQR 20-34), significantly older among those with reinfection (35 years [IQR 30-46] vs 25 years [IQR 20-32]; p=0.013). Median duration of injection drug use at enrolment was 5.5 years (IQR 2.2, 11.0), with shorter duration of injection drug use among those with reinfection (2.8 years [IQR 0.5, 5.2] vs 6.2 years [IQR 2.6, 12.5]; p=0.046). In unadjusted analysis, HCV reinfection in PWID was associated with methamphetamine injecting during follow up (p=0.010) and use of unsterile needles and/or syringes during follow up (p=0.002). On multivariate analysis, reinfection was associated with older age (aIRR 23.26, 95% CI 2.49, 319.35, p=0.003), shorter duration of injection drug use (duration >5.5 years: aIRR 0.05, 95% CI 0.00, 0.59; p=0.010) and use of unsterile needles and/or syringes during follow up (aIRR 43.27, 95%CI 5.52, 368.14, p<0.001) (Supplementary Table 4-6). Among HIV-positive MSM, injection drug use at end of and/or post-treatment was reported by 23% (ever injection drug use 61%); this was the only factor associated with HCV reinfection in this sub-group (aIRR 8.19, 95%CI 1.34, 85.99, p=0.019; adjusted for age) (Supplementary Table 4-7).

Page 152 of 309 Table 4-4. Factors associated with reinfection following treatment for recent HCV infection – Exact Poisson regression analysis

HCV No reinfection reinfection IRR 95% CI P aIRR 95% CI P

N=10 N=110 Sex, n (%) *

Male 10 (100) 90 (82) 1.00 - Female 0 19 (17) 0.37 0.00, 2.28 0.336 Transgender 0 1 (1) 3.35 0.00, 20.82 1.000 Age category (divided at median), n (%) ≤36 2 (20) 54 (49) 1.00 - 1.00 - >36 8 (80) 56 (51) 3.72 0.74, 35.99 0.137 5.42 1.06, 52.93 0.040 Social functioning score at enrolment, n (%) * ≤11 2 (20) 58 (53) 1.00 - >11 7 (70) 45 (41) 4.60 0.88, 45.34 0.079 Missing 1 (10) 7 (6) 3.06 0.05, 58.77 0.730 Mode of primary HCV acquisition, n (%) IDU 6 (60) 60 (55) 1.00 - Sexual 4 (40) 47 (43) 0.98 0.20, 4.14 1.000 Other 0 3 (3) 3.66 0.00, 25.36 1.000 HIV infection, n (%)

No 3 (30) 53 (48) 1.00 - Yes 7 (70) 57 (52) 2.31 0.53, 13.86 0.351 Injecting drug use ever at enrolment, n (%) No 3 (30) 33 (30) 1.00 - Yes 7 (70) 77 (70) 1.04 0.24, 6.26 1.000 Injection drug use in previous 6 months at enrolment, n (%) * No 4(40) 55 (50) 1.00 - Yes 6 (60) 53 (48) 1.40 0.33, 6.76 0.838 Missing 0 2 (2) 5.98 0.00, 47.84 1.000 Injection drug use in previous 30 days at enrolment, n (%) No 4 (40) 65 (59) 1.00 - Yes 6 (60) 45 (41) 1.83 0.44, 8.84 0.522 IDU at end of treatment and/or post treatment, n (%) ** ** No 2 (20) 70 (64) 1.00 - 1.00 - Yes 8 (80) 37 (34) 5.88 1.17, 56.82 0.027 7.86 1.54, 76.79 0.008 Missing 0 3 (3) 5.51 0.00, 71.51 1.000 5.78 0.00, 74.36 1.000 P overall for categorical variables: * ≥0.05, ** 0.001-0.05, *** <0.001 Univariate analysis - P overall: Sex, p=0.281, social functioning score, p=0.079, IDU in previous 6 months, p=0.838, IDU at end of and/or post treatment, p=0.027 Multivariate analysis – P overall: IDU at end of and/or post treatment, p=0.008

Page 153 of 309 Discussion This analysis assessed HCV reinfection incidence amongst individuals treated for recent HCV infection (duration of infection <18 months) who achieved an end-of-treatment response. High levels of risk behaviour associated with HCV transmission were reported, including 38% reporting injection drug use at end of and/or post treatment, predominantly methamphetamine. Ten cases of reinfection were identified for an overall reinfection incidence of 7.4 per 100 py. All cases occurred in men, the majority of whom were HIV-positive MSM (n=7) and reported injection drug use at end of and/or post treatment (n=8). Two cases occurred in HIV-positive MSM who denied ever injecting drugs.

The incidence of reinfection following treatment for recent HCV infection reported in this analysis is consistent with previous studies amongst HIV-positive MSM and PWID (reinfection incidence: 9.6 – 15.2 per 100 py) (12, 269, 270), and expands upon the previous analysis limited to the predominantly HCV mono-infected ATAHC I cohort (285). The higher incidence of HCV reinfection in this and other acute cohorts contrasts with the majority of published studies in individuals treated for chronic HCV infection (summarised in Supplementary Table 4-1). In a recent meta-analysis, Simmons et al (268) examined the risk of HCV recurrence following interferon-based treatment-induced SVR in three different populations, defined by their perceived risk of reinfection – HCV mono-infected “low risk” (no recognised risk factors for reinfection), HCV mono-infected “high risk” (former or recent injection drug use, incarceration, MSM) and HIV/HCV co-infection. Reinfection incidence was 0.0 per 100 py (95% 0.0, 0.0) in those deemed “low risk”, 1.9 per 100 py (95% CI 1.1, 2.8) in those deemed “high risk” and 3.2 per 100 py (95% CI 0.0, 12.3) in those with HIV/HCV co-infection. However, the proportion of “high risk” or HIV/HCV co-infected individuals continuing to engage in behaviours which facilitated HCV transmission and placed them at risk of reinfection was unclear.

When assessing suitability for HCV therapy, certain populations, including PWID and people with HIV/HCV co-infection, have been considered “high-risk”, primarily based on the apparent potential for reinfection (268). However, these populations are heterogeneous with different levels of risk attributable to specific subgroups. Sub-populations of PWID include those who report injecting an illicit drug at least once (lifetime PWID), those who have ceased injecting drug use (former PWID) and those who continue to inject drugs (recent PWID, with definitions of “recent” varying between one to 12 months) (272). Understanding the definitions for different PWID populations is crucial to accurately define reinfection risk following therapy. Similarly, not all people with HIV/HCV co-infection demonstrate contemporary behaviours placing them at risk of reinfection. While the internationally observed increase in HCV

Page 154 of 309 incidence in HIV-positive MSM has been associated with sexual risk behaviour and recreational drug use (12), as with primary HCV infection (11, 57), HIV-positive MSM who inject drugs are at significantly higher risk of HCV reinfection than HIV-positive MSM who do not inject drugs. As exemplified in this cohort, populations at high risk of reinfection, such as PWID and HIV- positive MSM, are not mutually exclusive.

The risk of HCV reinfection following treatment is significantly higher in those who report ongoing behaviour facilitating HCV transmission, with reinfection incidence ranging between 0.0 – 33.0 per 100 py in PWID treated for chronic HCV infection who reported ongoing injection drug use (246, 274, 275, 281, 285-288). Similarly, in this cohort, HCV reinfection incidence was significantly higher amongst participants who reported injection drug use during follow up as compared with those who did not. However, reinfection was not associated with injection drug use prior to or at commencement of therapy. Particularly in the setting of interferon-based therapy, there may have been considerable selection bias in those PWID deemed suitable, or willing, for treatment. While injecting risk behaviour among PWID appeared to decline during and after interferon-based treatment (338), it is possible that expanded HCV treatment access and DAA therapeutic optimism may be associated with increased risk behaviour, as seen among MSM following the introduction of HIV combination antiretroviral therapy (339).

Reinfection was associated with injection drug use following treatment and older age, the latter appearing to be related to older age and shorter duration of injection drug use among PWID with reinfection. Recent onset of injection drug use has been associated with HCV acquisition, though typically among young PWID (9, 10, 39-43). The increased risk of reinfection seen with injection drug use post treatment, use of unsterile needles and syringes and more recent onset of injection drug use highlights the need for education and broad access to harm reduction and prevention strategies in concert with HCV treatment. For PWID, access to interventions known to prevent HCV infection, including OST and high coverage needle and syringe access programs (40, 47-49, 340), will be crucial.

However, differences in drug use and sexual behaviours among cohorts of HIV-positive MSM as compared with HCV mono-infected populations may necessitate different public health strategies. Much of the literature surrounding HCV acquisition and prevention among PWID focusses on individuals who inject opiates (40, 47-49, 340). Older age at injecting onset, increasing use of stimulant drugs (largely amphetamines) and the phenomena of ‘chemsex’ (illicit drug use before or during sex, by both injecting and non-injecting routes of administration) may necessitate a different approach in MSM (77, 80-83). Evidence supporting sexual behavioural interventions for HCV prevention among MSM is lacking. With serosorting Page 155 of 309 of sexual partners by HIV-status and increasing use of pre-exposure prophylaxis to prevent HIV transmission in HIV-negative MSM, there is the potential for increased sexual risk behaviour and transmission of HCV among MSM populations (77, 86, 87). With DAA treatment scale-up among traditionally marginalised or “high-risk” populations, implementation and evaluation of novel prevention strategies should be a priority.

This study has a number of strengths, including the prospective design, inclusion of active PWID and HIV/HCV co-infected MSM, robust definition of follow up time at-risk for reinfection and use of viral sequencing to accurately delineate relapse and reinfection. The inclusion of a relatively large at-risk population, and documentation of ten cases of reinfection, provided sufficient power to evaluate associations. The prospective design allowed for serial HCV RNA measurements, improving the accuracy of the date of HCV reinfection estimation. Time at-risk for reinfection was calculated from date of end of treatment, where previous analyses have calculated time at-risk from date of SVR. In the era of DAA therapy, reinfection incidence rates will need to be calculated from end of treatment and sequencing used to accurately determine the aetiology of post-treatment HCV RNA recurrence to avoid misclassification, with reinfection occurring prior to the primary endpoint (SVR12) also seen in DAA registration trials (239, 251). However, the sequencing methodology used in this analysis could be considered suboptimal, given the inherent limitations of population-based (Sanger) sequencing, including poor sensitivity to detect minor variants and inability to detect mixed infection (266). Use of next-generation sequencing (NGS) could provide additional clarity in classification of post-treatment viral recurrence.

There are other limitations to this study. Firstly, duration of follow up was limited to that stipulated in the original trial protocol, ranging from 48 to 120 weeks post treatment. Two cases of confirmed reinfection were of indeterminate outcome as reinfection occurred at the last study visit. Given the follow up time, it is also possible that some participants were not followed for a sufficient time to allow for spontaneous clearance of reinfection. Additionally, while the cohort at-risk for reinfection was sizeable, the total follow up time post treatment was impacted by short individual follow up, which could bias reinfection incidence, by creating a “cohort effect” in which those individuals at very high risk are reinfected early, while overall risk reduces over time. Secondly, due to the intervals between HCV RNA tests (12-24 weeks during follow up), some reinfections with rapid clearance may have been missed and as such, the reported reinfection incidence is an underestimate (341). However, this would not have impacted detection of persistent HCV reinfection. Thirdly, sexual behaviour was not collected, and as such, this could not be included in the model. However, the association between ongoing infecting drug use and reinfection in HIV-positive MSM highlights the overlap in these populations. Finally, only six percent of the cohort received an interferon-free DAA regimen.

Page 156 of 309 Data is being to emerge on reinfection following treatment of chronic HCV infection with DAA therapy. In the C-EDGE COSTAR trial among people receiving OST, six cases of reinfection were identified at or prior to post treatment week 24, with five cases of reinfection detected at post treatment week eight (reinfection incidence 4.6 per 100 py, 95% CI 1.7, 10.0) (251). Urine drug screen was positive both during and following treatment in five of the six cases. The incidence of reinfection following DAA-based treatment needs careful evaluation as access to treatment among populations at-risk of ongoing transmission increases.

The treatment paradigm for individuals with HCV infection is evolving rapidly (228-230, 239). The potential for broad access to highly effective, well tolerated interferon-free DAA regimens has stimulated discussion around HCV treatment-as-prevention. HCV treatment-as-prevention strategies will be enhanced by early diagnosis and increased treatment uptake in recent HCV infection, in order to reduce transmission amongst at-risk populations (75, 291). The significant risk for HCV reinfection following treatment in individuals with ongoing high risk behaviour emphasises the need for post-treatment surveillance, harm reduction strategies and education (270, 311), but must not be considered an impediment to treatment, if HCV elimination is to be achieved.

Page 157 of 309 Supplementary Material

Methods HCV RNA testing and sequencing

The presence of HCV RNA was assessed at all scheduled study visits. Specifically in regard to assessment for viral recurrence, HCV RNA testing was performed at end of treatment (including early treatment discontinuation visits) and at post treatment weeks 12, 24 and 48. Depending on the study protocol, HCV RNA testing was additionally performed at the following post treatment time points: week four (DARE-C I, DARE-C II), 36 (ATAHC I), 60 (ATAHC I), 72 (ATAHC I, ATAHC II, DARE-C I), 84 (ATAHC I), 96 (ATAHC I), 108 (ATAHC I) and 120 (ATAHC I). In ATAHC I, HCV RNA was assessed using a qualitative HCV RNA assay (Versant transcription-mediated amplification [TMA]; Bayer, Australia; LLoD 10 IU/ml) and if positive, a quantitative HCV RNA assay (Versant HCV RNA 3.0; Bayer, Australia; LLoD 615 IU/ml). In ATAHC II, DARE-C I and DARE-C II, HCV RNA was assessed using a quantitative HCV RNA assay (COBAS Taqman 2.0; Roche Diagnostics, USA; LLoD 15 IU/mL).

HCV RNA sequencing was performed on the first available pre-treatment quantifiable HCV RNA sample and the first available quantifiable HCV RNA sample following HCV RNA recurrence with methods described previously (302, 303). In brief, sequencing was attempted on all samples with quantifiable HCV RNA. HCV RNA was extracted from 140 μL of plasma using QIAamp viral extraction mini kit (Qiagen) according to manufacturers’ instructions and eluted in 80 μl buffer. Reverse transcription was performed with random hexamers using the superscript VILO cDNA synthesizer kit (Life Technologies), containing 5 μL RNA, 1 μL Superscript Enzyme Mix, 2 μL VILO reaction Mix and 2 μL of PCR additive PolyMate (Bioline, UK). Complementary DNA was generated using SuperScript VILO cDNA Synthesis Kit (Life Technologies, Carlsbad, CA) with random hexamers. A 1,514 bp fragment of the HCV genome covering Core, Envelope-1, hypervariable region-1, and beginning of Envelope-2 (E2) was amplified. NS5B (388bp) was amplified by a single round PCR with some modifications to reaction conditions (303). Samples not successfully amplified were retested with a modified reverse transcription methodology, whereby the reaction was performed at a lower temperature, for a longer duration without the addition of the PolyMate Additive. Purified amplicons were sequenced using the Sanger method. The genotype was determined for all subjects for both Core-E2 and NS5B sequences using the Oxford HCV Automated Subtyping Tool (www.bioafrica.net/rega-genotype/html/subtypinghcv.html).

Page 158 of 309

Statistical analysis

In the cohort at-risk for reinfection (ETR without post-treatment relapse), categorical parameters were summarised as number and proportion and continuous variables were summarised by either mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. Reinfection incidence was calculated using person-time of observation. Confidence intervals (CI) for rates were calculated using Poisson distribution.

Exact Poisson regression analysis was used to assess factors associated with HCV reinfection, with time at risk (years) as the exposure variable. In unadjusted analyses, potential predictors were determined a priori and included sex, age at study enrolment, income, education level, social functioning score at enrolment (median), mode of HCV acquisition (injection drug use, sexual, other), HIV infection, injection drug use (ever, previous 6 months at enrolment, previous 30 days at enrolment), and injection drug use at end of and/or post-treatment. Social functioning was calculated using a validated scale from the Opiate Treatment Index (313) and addressed employment, residential stability, interpersonal conflict and social support (higher score reflects poorer social functioning, range score 0-48). A second model assessed factors associated with reinfection among lifetime PWID. Potential predictors included sex, age at commencing injection drug use, social functioning score at enrolment (median), HIV infection, injecting frequency at enrolment (none, less than daily, daily or more), methamphetamine injecting at end of and/or post treatment and use of unsterile needles and/or syringes at end of and/or post treatment. A third model assessed factors associated with reinfection among HIV- positive MSM. Potential predictors included sex, age at study enrolment, social functioning score at enrolment (median), mode of HCV acquisition (injection drug use, sexual, other), injection drug use (ever, previous 6 months at enrolment, previous 30 days at enrolment), injection drug use at end of and/or post-treatment, and methamphetamine injecting at end of and/or post treatment. All variables with p<0.2 in univariate analysis were considered in multivariate regression models using a backwards stepwise approach. Statistically significant differences were assessed at p<0.05; p-values were two-sided. Analysis was performed using STATA version 14.0 (StataCorp, College Station, TX).

Page 159 of 309 Tables Supplementary Table 4-1. HCV reinfection incidence following treatment induced viral clearance (2011 – 2016) Reinfection Number Location and Number of incidence Author, year Population of study design reinfections per 100 py subjects (95% CI) Meta-analysis studies: Reinfection post treatment for acute and chronic HCV infection (268) Simmons Meta-analysis 0.0 “Low risk” 1 7969 4 2016 (31 studies) (0.0, 0.0) (268) Simmons “High risk”: PWID, Meta-analysis 1.9 771 36 2016 incarcerated 1 (14 studies) (1.1, 2.8) (268) Simmons Meta-analysis 3.2 HIV/HCV coinfection 1 309 31 2016 (4 studies) (0.0, 12.3) (12) Hagan Meta-analysis 11.4 HIV-positive MSM 170 38 2015 (2 studies) (7.4, 17.7) (246) Aspinall Meta-analysis 2.4 PWUD 131 7 2013 (5 studies) (0.9, 6.1) Primary studies: Reinfection post treatment for chronic HCV infection (251) Dore OST (100%) International 4.6 301 6 2016 Recent PWUD (58%) Prospective (1.7, 10.0) (274) Midgard Lifetime PWID (100%) Norway 2.0 94 12 2016 Recent PWID (39%) Prospective (1.0, 3.5) (275) Weir Scotland 1.7 Lifetime PWID (100%) 277 7 2016 Retrospective (0.7, 3.5) (276) Pineda HIV-positive (100%) Spain 1.2 84 4 2015 Lifetime PWID (86%) Prospective (0.3, 3.1) (277) Conway * Canada 2.9 Recent PWID (100%) 70 4 2013 Prospective (1.1, 7.2) (278) Deshaies * Canada 6.3 Recent PWID (100%) 20 2 2013 Prospective (1.7, 20.3) (279) Edlin * US 2.2 Recent PWID (100%) 15 1 2013 Not reported (3.9, 11.5) (280) Hilsden * Canada 2.8 Recent PWUD (100%) 23 1 2013 Prospective (0.0, 14.5) Incarcerated (100%) (281) Marco * Spain Recent PWID (10%) 119 9 5.3 2013 Retrospective HIV-positive (15%) Serbia (282) Ruzic * 0 Former PWID (100%) Retrospective - 20 0 2013 (0.0, 3.7) prospective (283) Grady * The Netherlands 0.8 Recent PWUD (100%) 42 1 2012 Prospective (0.0, 3.7) (284)Manolakopoulos* Lifetime PWID (100%) Greece 4.1 61 5 2012 Recent PWID (57%) Retrospective (1.8, 9.2) Primary studies: Reinfection post treatment for recent HCV infection (269) Lambers ** The Netherlands 15.2 HIV-positive MSM (100%) 56 11 2011 Retrospective (8.0, 26.5) (285) Grebely PWID Australia 12.3 67 5 2012 HIV-positive MSM Prospective (5.1, 29.6) (270) Martin * ** England 9.6 HIV-positive MSM (100%) 1142 27 2013 Retrospective (6.6, 14.1) *Studies included in meta-analysis performed by Simmons et al (268) ** Studies included in meta-analysis performed by Hagan et al (12)

1 Simmons et al (268) examined the risk of HCV recurrence following SVR in three different populations, defined by their risk of reinfection – HCV mono-infected “low risk” (no recognised risk factors for reinfection), HCV mono- infected “high risk” (recognised risk factors for reinfection: former or recent injecting drug use [12 studies] incarceration [2 studies], MSM [0 studies]) and HIV/HCV co-infection. 2 Documented primary HCV infection only

Abbreviations: MSM, men-who-have-sex-with-men; OST, opiate substitution therapy; PWID, people who inject drugs; PWUD, people who use drug.

Page 160 of 309 Supplementary Table 4-2. Drug use characteristics amongst participants at-risk for HCV reinfection Overall Reinfection No reinfection Drug use characteristics N=120 N=10 N=110 IDU, n (%) Lifetime 84 (70)* 8 (80) 76 (69) Ever prior to enrolment 83 (69)* 7 (70) 77 (69) Previous 6 months prior to enrolment 59 (49) 6 (60) 53 (48) Previous 30 days prior to enrolment 51 (43) 6 (60) 45 (41) At end of or post treatment 45 (38) 8 (80) 37 (34) Amongst PWID: N=84 N=8 N=76 Age at first IDU, median (IQR) 25 (20-34) 35 (30-46) 25 (20-32) Duration of IDU at enrolment (years), 5.6 (2.2, 11.0) 2.8 (0.5, 5.2) 6.2 (2.5, 12.5) median (IQR) IDU frequency within 30 days of enrolment, n (%) None 31 (37) 2 (25) 29 (38) Less than daily 39 (46) 3 (38) 36 (47) Daily or more 12 (14) 3 (38) 9 (12) Unknown 2 (2) 0 2 (3) Peak IDU frequency at end of or post treatment None 37 (44) 0 37 (49) Less than daily 32 (38) 6 (75) 26 (34) Daily or more 12 (14) 2 (25) 10 (13) Unknown 3 (4) 0 3 (4) Amphetamine IDU at end of or post 32 (38) 7 (88) 25 (33) treatment Use of unsterile or shared needle, syringe or equipment during follow up No 64 (76) 2 (25) 63 (83) Yes 15 (18) 6 (75) 9 (12) Missing 5 (6) 0 5 (7) Use of unsterile needles and/or syringes during follow up No 69 (82) 3 (38) 66 (87) Yes 10 (12) 5 (63) 5 (7) Missing 5 (6) 0 5 (7) Use of shared injecting equipment during follow up No 70 (83) 5 (62) 65 (86) Yes 9 (11) 3 (38) 6 (8) Missing 5 (6) 0 5 (7) Amongst PWID reporting IDU N=51 N=6 N=45 within 30 days of enrolment: Drug most often injected Amphetamines 31 (61) 3 (50) 28 (62) Heroin and other opiates 15 (29) 1 (17) 14 (31) Other 4 (8) 2 (33) 2 (4) Unknown 1 (2) 0 1 (2) *One participant reported first injection drug use post treatment

Page 161 of 309 Supplementary Table 4-3. Drug use characteristics amongst participants at-risk for HCV reinfection by HIV serostatus HCV mono- HIV/HCV Drug use characteristics infection P N=64 N=56 IDU, n (%) Lifetime 45 (80) 39 (61)* 0.021 Ever prior to enrolment 45 (80) 38 (59)* 0.013 Previous 6 months prior to enrolment 34 (61) 25 (39) 0.035 Previous 30 days prior to enrolment 32 (57) 19 (30) 0.002 At end of or post treatment 31 (55) 14 (22) <0.001 Age at first IDU, median (IQR) 23 (18-30) 30 (25-41) <0.001 Amongst PWID: N=45 N=39 IDU frequency within 30 days of enrolment, n (%) None 11 (24) 20 (51) 0.001 Less than daily 20 (44) 19 (49) Daily or more 12 (27) 0 Unknown 2 (4) 0 Peak IDU frequency at end of or post treatment, n (%) None 14 (31) 23 (59) 0.012 Less than daily 18 (40) 14 (36) Daily or more 11 (24) 1 (3) Unknown 2 (4) 1 (3) Amphetamine IDU at end of or post treatment, n (%) 17 (38) 14 (36) 0.859 Use of unsterile or shared needle, syringe or equipment during follow up, n (%) No 30 (67) 34 (87) 0.086 Yes 11 (24) 4 (10) Missing 4 (9) 1 (3) Use of unsterile needles and/or syringes during follow up, n (%) No 33 (73) 36 (92) 0.077 Yes 8 (18) 2 (5) Missing 4 (9) 1 (3) Use of shared injecting equipment during follow up, n (%) No 36 (80) 34 (87) 0.461 Yes 5 (11) 4 (10) Missing 4 (9) 1 (3) Amongst PWID reporting IDU within 30 days of N=32 N=19 enrolment: Drug most often injected, n (%) Amphetamines 13 (41) 18 (95) 0.002 Heroin and other opiates 14 (44) 1 (5) Other 4 (13) 1 (5) Unknown 1 (3) 0 *One participant reported first injection drug use post treatment

Page 162 of 309 Supplementary Table 4-4. Incidence of HCV reinfection among PWID treated for recent HCV infection

Cases of Incidence Participants Participant type reinfection PYFU per 100 95% CI at risk (n) (n) py IDU ever at enrolment

Confirmed/possible reinfection 7 83 93 7.5 3.6, 15.8 Confirmed reinfection 6 83 93 6.4 3.0, 14.3 Confirmed persistent reinfection 4 83 93 4.3 1.6, 11.4

IDU within six months of enrolment

Confirmed/possible reinfection 6 59 69 8.7 3.9, 19.5 Confirmed reinfection 5 59 69 7.3 3.0, 17.5 Confirmed persistent reinfection 4 59 69 5.8 2.2, 15.5

IDU within 30 days of enrolment

Confirmed/possible reinfection 6 51 61 9.9 4.4, 22.0 Confirmed reinfection 5 51 61 8.2 3.4, 19.8 Confirmed persistent reinfection 4 51 61 6.6 2.5, 17.6

IDU at end of and/or post-treatment Confirmed/possible reinfection 8 45 52 15.3 7.7, 30.6 Confirmed reinfection 6 45 52 11.5 5.2, 25.5 Confirmed persistent reinfection 4 45 52 7.7 2.9, 20.4 Abbreviations: CI, confidence interval; IDU, injecting drug use; py, person years; PYFU, total person years follow up

Page 163 of 309 Supplementary Table 4-5. Factors associated with confirmed reinfection following treatment for recent HCV infection – Exact Poisson regression analysis

HCV No aIR reinfection reinfection IRR 95% CI P 95% CI P R N=8 N=110 Sex, n (%) *

Male 8 (100) 90 (82) 1.00 - - Female 0 19 (17) 0.46 0.00, 2.95 0.469 Transgender 0 1 (1) 4.15 0.00, 26.89 1.000 Age category (divided at median), n (%) ≤36 1 (13) 54 (49) 1.00 - - 1.00 - - >36 7 (87) 56 (51) 6.57 0.84, 296.18 0.085 9.39 1.18, 427.98 0.028 Social functioning score at enrolment, n (%) * ≤11 2 (25) 58 (53) 1.00 - - >11 5 (63) 45 (41) 3.40 0.56, 35.69 0.242 Missing 1 (13) 7 (6) 3.06 0.05, 58.77 0.730 Mode of primary HCV acquisition, n (%) * IDU 5 (63) 60 (55) 1.00 - - Sexual 3 (38) 47 (43) 0.90 0.12, 4.61 1.000 Other 0 3 (3) 4.41 0.00, 32.34 1.000 HIV infection, n (%) No 2 (25) 53 (48) 1.00 - - Yes 6 (75) 57 (52) 3.00 0.54, 30.42 0.289 Injecting drug use ever at enrolment, n (%) No 2 (25) 35 (31) 1.00 - - Yes 6 (75) 77 (69) 1.31 0.23, 13.29 1.000 Injection drug use in previous 6 months at enrolment, n (%) * No 3 (38) 55 (50) 1.00 - - Yes 5 (62) 53 (48) 1.54 0.30, 9.93 0.813 Missing 0 2 (2) 8.05 0.00, 74.97 1.000 Injection drug use in previous 30 days at enrolment, n (%) No 3 (38) 65 (59) 1.00 - - Yes 5 (62) 45 (41) 2.02 0.39, 13.04 0.527 IDU at end of treatment and/or post treatment, n (%) * ** No 2 (25) 70 (64) 1.00 - - 1.00 - - Yes 6 (75) 37 (34) 4.68 0.84, 47.45 0.088 6.60 1.16, 67.42 0.030 Missing 0 3 (3) 5.56 0.00, 71.51 1.000 5.83 0.00, 75.00 1.000 P overall for categorical variables: * ≥0.05, ** 0.001-0.05, *** <0.001 Univariate analysis - P overall: Sex, p=0.281, social functioning score, p=0.079, IDU in previous 6 months, p=0.838, IDU at end of and/or post treatment, p=0.027 Multivariate analysis – P overall: IDU at end of and/or post treatment, p=0.008

Page 164 of 309 Supplementary Table 4-6. Factors associated with reinfection following treatment for recent HCV infection amongst PWID – Exact Poisson regression analysis

HCV No reinfection reinfection IRR 95% CI P aIRR 95% CI P

N=8 N=76 Age category (divided at median), n (%) ≤36 2 (25) 44 (58) 1.00 - - 1.00 - - >36 6 (75) 32 (42) 3.68 0.66, 37.30 0.175 23.26 2.49, 319.35 0.003 Social functioning score at enrolment, n (%) ≤11 2 (25) 39 (51) 1.00 - - >11 6 (75) 32 (42) 3.64 0.65, 36.92 0.178 Missing 0 5 (7) 2.49 0.00, 31.97 1.000 HIV infection No 3 (38) 42 (55) 1.00 - - Yes 5 (63) 34 (45) 2.18 0.42, 14.05 0.458 Duration of IDU at enrolment (divided at median), n (%) * ** ≤5.5 years 6 (75) 34 (45) 1.00 - - 1.00 - - >5.5 years 1 (13) 40 (53) 0.16 0.00, 1.28 0.103 0.05 0.00, 0.59 0.010 Missing 1 (13) 2 (3) 2.25 0.05, 18.55 0.787 0.10 0.00, 1.53 0.121 IDU in previous 6 months at enrolment, n (%) * No 2 (25) 22 (29) 1.00 - - Yes 6 (75) 53 (70) 1.06 0.19, 10.70 1.000 Missing 0 1 (1) 6.29 0.00, 80.85 1.000 Injecting frequency within 30 days of enrolment, n (%) * None 2 (25) 29 (38) 1.00 - - < daily 3 (38) 36 (47) 0.92 0.11, 11.05 0.270 ≥ daily 3 (38) 9 (12) 3.90 0.45, 46.72 1.000 Missing 0 2 (3) 3.53 0.00, 45.32 1.000 Methamphetamine injecting during follow-up, n (%) No 1 (13) 52 (68) 1.00 - - 1.53, Yes 7 (88) 24 (32) 11.94 0.010 538.29 Use of unsterile needles and/or syringes during follow up, n ** *** (%) No 3 (38) 66 (87) 1.00 1.00 - - Yes 5 (63) 5 (7) 13.19 2.57, 84.93 0.002 43.27 5.52, 368.14 <0.001 Missing 0 5 (7) 2.46 0.00, 22.90 1.000 3.42 0.00, 31.86 1.000 P overall for categorical variables: * ≥0.05, ** 0.001-0.05, *** <0.001

Univariate analysis - P overall: Social functioning score, p=0.178; IDU in previous 6 months, p=1.000; injecting frequency at enrolment, p=0.270; use of unsterile needles and/or syringes during follow up, p=0.002

Multivariate analysis – P overall: Duration of IDU at enrolment, p=0.010; use of unsterile needles and/or syringes during follow up, p<0.001

Page 165 of 309 Supplementary Table 4-7. Factors associated with reinfection following treatment for recent HCV infection amongst HIV-positive MSM – Exact Poisson regression analysis

HCV No reinfection reinfection IRR 95% CI P aIRR 95% CI P

N=7 N=57 Age category (divided at median), n (%) ≤36 0 19 (33) 1.00 - - 1.00 >36 7 (100) 38 (67) 4.37 0.66, inf 0.145 5.69 0.85, inf 0.078 Social functioning score at enrolment, n (%) * ≤11 2 (29) 35 (61) 1.00 - - >11 4 (57) 19 (33) 4.07 0.58, 44.99 0.193 Missing 1 (14) 3 (5) 4.23 0.07, 81.29 0.569 Mode of primary HCV acquisition, n (%) IDU 3 (43) 19 (33) 1.00 - - Sexual 4 (57) 38 (67) 0.79 0.13, 5.37 1.000 IDU ever at enrolment, n (%) No 3 (43) 23 (40) 1.00 - - Yes 4 (57) 34 (60) 0.94 0.16, 6.44 1.000 IDU in previous 6 months at enrolment, n (%) * No 4 (57) 33 (58) 1.00 - - Yes 3 (43) 22 (39) 1.21 0.18, 7.19 1.000 Missing 0 2 (4) 3.78 0.00, 30.29 1.000 IDU in previous 30 days at enrolment, n (%) No 4 (57) 41 (72) 1.00 - - Yes 3 (43) 16 (28) 1.80 0.26, 10.63 0.681 IDU at end of treatment and/or post treatment, n (%) ** ** No 2 (29) 46 (81) 1.00 - - 1.00 - - Yes 5 (71) 10 (18) 6.87 1.12, 72.11 0.035 8.19 1.34, 85.99 0.019 Missing 0 1 (2) 21.03 0.00, 270.30 1.000 15.01 0.00, 192.92 1.000 Methamphetamine injecting during follow-up, n (%) No 3 (43) 47 (82) 1.00 - - Yes 4 (57) 10 (18) 4.11 0.70, 28.06 0.132 P overall for categorical variables: * ≥0.05, ** 0.001-0.05, *** <0.001

Univariate analysis - P overall: Social functioning score, p=0.193, IDU in previous 6 months, p=1.000, IDU at end of treatment and/or post treatment, p=0.035 Multivariate analysis – P overall: IDU at end of treatment and/or post treatment, p=0.019

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Figures 1.00 0.75 0.50 0.25 0.00 0 .5 1 1.5 2 2.5 Time from end of treatment (years) Number at risk 120 96 64 30 7 1

Supplementary Figure 4-1. Kaplan-Meir curve of the probability of HCV reinfection following treatment for recent HCV infection

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Chapter 5 Optimising direct-acting antiviral regimen choice in HIV/HCV co- infection – informing future trial design

Chapter Introduction and Objectives The availability of interferon-free direct-acting antiviral regimens for HCV should diminish barriers to therapy in HIV/HCV co-infection. However, treatment of HIV/HCV co-infected individuals will require an awareness of the potential drug-drug interactions between specific direct-acting antivirals and HIV antiretroviral agents in order to prevent morbidity and ensure treatment efficacy. Similarly, in designing future pragmatic clinical trials in recent HCV infection, direct-acting antiviral regimen choice should be optimised.

In this real-world cohort of HIV/HCV co-infected individuals in Sydney, Australia, a simulation of potential drug-drug interactions between participants’ combination antiretroviral therapy and interferon-free direct-acting antiviral regimens was performed using www.hep- druginteractions.org and relevant prescribing information. The combinations of sofosbuvir plus an NS5A inhibitor and sofosbuvir plus ribavirin appeared to be largely suitable for co- administration with commonly prescribed combination antiretroviral therapy, while drug-drug interactions frequently complicated use of HCV NS3/4a protease inhibitor-containing regimens. However, despite minimal drug-drug interactions, the combination of sofosbuvir plus ribavirin has been rendered largely obsolete, given suboptimal efficacy. In optimising direct-acting antiviral regimen choice for future trial design in recent HCV infection, the evaluation of a short duration strategy involving sofosbuvir plus an NS5A inhibitor appears to be most appropriate, given favourable characteristics, including potency and minimal drug-drug interactions.

The manuscript has been published in Open Forum Infectious Diseases.

Publication Martinello M, Dore GJ, Skurowski J, Bopage RI, Finlayson R, Baker D, Bloch M, Matthews GV. Antiretroviral use in the CEASE cohort study and implications for DAA therapy in HIV/HCV co-infection. Open Forum Infectious Diseases. 2016 May 18;3(2): ofw105. doi: 10.1093/ofid/ofw105.

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Declaration I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Marianne Martinello

Viral Hepatitis Clinical Research Program Kirby Institute, UNSW Australia Wallace Wurth Building, Sydney NSW 2052 t: +61 413 276 968 e: [email protected]

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Co-authorship Acknowledgement In the case of Chapter Five, the nature and extent of my contribution to the work was the following:

Author name Contribution (%) Nature of contribution

Contributed to study design, conducted the data analysis, contributed to data collection and led Marianne Martinello 55 the development, writing and critical revision of the manuscript

Contributed to study design, data collection, Gregory J Dore 15 interpretation of findings and critical revision of the manuscript

Coordinated implementation of the study and Jasmine Skurowski 3 data collection. Contributed to revision of the manuscript.

Contributed to study design, implementation, Rohan I Bopage 3 data collection and revision of the manuscript

Contributed to study design, implementation, Robert Finlayson 3 data collection and revision of the manuscript

Contributed to study design, implementation, David Baker 3 data collection and revision of the manuscript

Contributed to study design, implementation, Mark Bloch 3 data collection and revision of the manuscript

Contributed to study design, data collection, Gail V Matthews 15 interpretation of findings and critical revision of the manuscript

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Antiretroviral use in the CEASE cohort study and implications for DAA therapy in HIV/HCV co-infection.

Marianne Martinello1,2, Gregory J Dore1,2, Jasmine Skurowski1, Rohan I Bopage3,4, Robert Finlayson5, David Baker6, Mark Bloch7, Gail V Matthews1,2

1. Viral Hepatitis Clinical Research Program, The Kirby Institute, UNSW Australia, Sydney, NSW, Australia 2. Department of Infectious Diseases and Immunology, St Vincent’s Hospital, Sydney, NSW, Australia 3. The Albion Centre, Sydney, NSW, Australia 4. School of Public Health and Community Medicine, UNSW, Australia, Sydney, NSW, Australia 5. Taylor Square Private Clinic, Sydney, NSW, Australia 6. East Sydney Doctors, Sydney, NSW, Australia 7. Holdsworth House Medical Practice, Sydney, NSW, Australia

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Abstract Background and Objectives: Interferon-free direct-acting antiviral (DAA) regimens for HCV provide a major advance in clinical management, including in HIV/HCV co-infection. Drug- drug interactions (DDI) with combination antiretroviral therapy (cART) will require consideration. This study aimed to characterise the cART regimens in HIV/HCV co-infected individuals and assess the clinical significance of DDIs with DAAs in a real-world cohort.

Methods: This analysis included participants enrolled in CEASE-D, a prospective cohort of HIV/HCV co-infected individuals in Sydney, Australia, between July 2014 and December 2015. A simulation of potential DDIs between participants’ cART and interferon-free DAA regimens was performed using www.hep-druginteractions.org and relevant prescribing information.

Results: In individuals on cART with HCV GT 1 and 4 (n=128), category 3 DDIs (contra- indicated or not recommended) were noted in 0% with sofosbuvir/ledipasvir, 0% with sofosbuvir plus daclatasvir, 17% with sofosbuvir/velpatasvir, 36% with ombitasvir/paritaprevir/ritonavir +/- dasabuvir, 51% with grazoprevir/elbasvir and 51% with sofosbuvir plus simeprevir; current cART regimens were suitable for co-administration in 100%, 100%, 73%, 64%, 49% and 49%, respectively. In individuals with HCV GT 2 or 3 (n=53), category 3 DDIs were evident in 0% with sofosbuvir plus daclatasvir, 0% with sofosbuvir and ribavirin and 13% with sofosbuvir/velpatasvir; current cART regimens were suitable in 100%, 100% and 81%, respectively.

Conclusions: Potential DDIs are expected and will impact on DAA prescribing in HIV/HCV co-infection. Sofosbuvir in combination with an NS5A inhibitor or ribavirin appeared to be the most suitable regimens in this cohort. Evaluation of potential DDIs is required to prevent adverse events or treatment failure.

Registration: ClinicalTrials.gov identifier NCT02102451

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Introduction The global burden of disease attributed to human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection is substantial with anti-HCV antibody prevalence estimated at 1.6 – 2.8% (2) and HIV antibody prevalence at 0.8% (342). Based on global HIV and HCV prevalence and estimates of the overlap in these epidemics, 2−5 million people are estimated to be co-infected with HIV and HCV (62, 343). The natural history of HIV and HCV are significantly impacted by the co-existence of the other virus with accelerated liver disease progression and increases in all-cause, AIDS-related and liver-related morbidity, hospitalisation and mortality, even in those people receiving combination antiretroviral therapy (cART) (63, 65, 66). While the number of deaths related to HIV is falling (342), the number of deaths attributed to HCV-related liver disease is rising (344).

Interferon-based HCV therapy has had limited success in HIV-positive populations, with concerns regarding efficacy and tolerability. While a sustained virological response (SVR) reduces both liver and non-liver related complications and mortality, therapy with pegylated- interferon and ribavirin resulted in SVR in less than 30% of HIV-positive individuals with HCV genotype 1 (134, 135).

The availability of interferon-free direct-acting antiviral (DAA) regimens for HCV offers considerable promise in the management of HIV/HCV co-infection (237-239, 243), with high efficacy, improved tolerability, shorter treatment duration and lower pill burden (140). However, in the context of concomitant cART, DDIs require consideration. To date, all approved DAAs demonstrate interactions with CYP450 enzymes or transporters, including P- glycoprotein (P-gp) and breast cancer resistance protein, with potential implications for DDIs (summarised in Supplementary Table 5-1). Safety data on potentially significant antiretroviral and DAA DDIs in HIV/HCV co-infected individuals are limited to the drug combinations permitted in phase II and III trials, with most trials having strict antiretroviral eligibility criteria (summarised in Supplementary Table 5-2). Data is emerging on the real-world relevance of DDIs in HCV-infected populations using interferon-free DAA therapy (345-348).

The aim of this analysis was to assess the clinical significance of DDIs between participants’ currently prescribed cART and interferon-free DAA regimens in a real-world HIV/HCV co- infected cohort.

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Methods Study design and participants

The Control and Elimination within Australia of Hepatitis C from people living with HIV (CEASE) Project is a prospective five year plan of enhanced HCV monitoring, primary care– based workforce development, rapid scale-up of HCV treatment and public health policy action in HIV-positive individuals within Australia. Data used in this analysis was collated from the first component of this project, CEASE-D: Surveillance of HCV, an ongoing prospective cohort study.

Enrolment in CEASE-D commenced in July 2014 at 5 sites in Sydney, New South Wales (tertiary hospital, n=1; primary care practice, n=4). The study population for this analysis included all individuals enrolled until December 2015 (n=257). HIV-positive participants were eligible for enrolment if they were 18 years of age or older and anti-HCV antibody positive. All participants were asked whether they would consider HCV therapy, both interferon-containing and interferon-free. Participants with detectable HCV RNA were considered for suitability of interferon-free DAA therapy. Further assessment of DDIs between cART and DAAs was based upon those with documented HCV genotype and cART regimen (Figure 5-1).

HCV GT 1, n=122

HCV GT 2, n=7

HCV GT 3, n=46 cART unknown, n=1 Detectable HCV RNA, n=208 HCV GT 4, n=6 Undetectable HCV RNA, On cART, n=249 n=32 HCV GT mixed, n=1 Missing, n=9 HCV GT indeterminate/unknown, n=26 Detectable HCV RNA, n=7 Enrolled, n=257 Not on cART, n=8 Missing, n=1

Figure 5-1. CEASE-D: Participant disposition

Abbreviations: cART, combination antiretroviral therapy; GT, genotype

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Assessment of liver disease

Initial laboratory assessments were conducted in concert with the participants’ standard-of-care with the presence of HCV RNA assessed using the COBAS Taqman HCV RNA assay, version 2.0 (lower limit of quantitation [LLoQ], 25 IU/mL; lower limit of detection [LLoD] 15 IU/mL; Roche Diagnostics, Branchburg, NJ, USA).

Fibrosis stage was graded by METAVIR classification, based on liver biopsy or transient elastography within 6 months of enrolment. For transient elastography, the following cut-off values were used: F0/F1, <7.1 kPa; F1/F2, ≥7.1 kPa; F2, ≥8.7 kPa; F3, ≥9.5 kPa; F3/F4, ≥12.5 kPa; and F4, ≥14.5 kPa (349, 350).

Assessment and classification of potential drug-drug interactions

The following approved and FDA-filed interferon-free DAA regimens were assessed: sofosbuvir/ledipasvir (351); ombitasvir/paritaprevir/ritonavir and dasabuvir (PrOD) (with and without ribavirin) (211); ombitasvir/paritaprevir/ritonavir (PrO) (with ribavirin) (211); grazoprevir/elbasvir (352); sofosbuvir plus simeprevir (217); sofosbuvir (210) plus daclatasvir (191); sofosbuvir plus ribavirin; and sofosbuvir/velpatasvir (240, 353, 354). Potential DDIs between the listed DAAs and documented antiretroviral drugs received by each individual were simulated according to the most recent literature, available prescribing information (as of April 2016) and the University of Liverpool DDI tool (www.hep-druginteractions.org). For each HCV genotype, DAA regimens chosen for analysis were based upon the 2015 EASL Clinical Practice Guidelines (103) and available prescribing information. As no participants in this cohort would have had additional significant DDIs related to ribavirin, DAA regimens with and without ribavirin were analysed together (in the case of PrOD +/- ribavirin for HCV GT 1a and 1b and PrO + ribavirin for GT4). The relationship and potential interaction between the DAA regimen and specific antiretroviral agents was designated as follows: category 1. no clinically significant DDI; category 2. potentially significant DDI – requiring additional monitoring for toxicity, adjustment of dose or timing of administration; category 3. co-administration not recommended or contra-indicated; or category 4. no data available. Category 2 included dose adjustment of daclatasvir and ritonavir-boosted HIV protease inhibitors. If a participant took more than 1 drug with different risks for a DDI, the highest category was chosen to determine the risk for that participant with a respective treatment regimen. Category 1 and 2 DDIs were considered suitable for co-administration of the DAA and cART regimen.

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Primary study endpoint

The primary study endpoint was the proportion of HIV/HCV co-infected individuals receiving suitable cART for co-administration with the above listed approved interferon-free DAA regimens.

Ethics and Study oversight

All study participants provided written informed consent before study procedures. The study protocol was approved by St Vincent’s Hospital, Sydney Human Research Ethics Committee (primary study committee), as well as by the institutional review board or independent ethics committee at each participating site and was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines and local regulatory requirements. The study was registered with ClinicalTrials.gov (NCT02102451).

Statistical analysis

Categorical parameters were summarised as number and proportion. Continuous variables were summarised by either mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. The number and proportion of individuals with DDIs category 1-4 was summarised by HCV genotype for each DAA regimen. Analysis was performed using STATA (version 14.0; StataCorp, College Station, TX).

Role of the Funding source

The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication are the views of the authors and do not necessarily represent the position of the Australian Government. Research reported in this publication was supported by Gilead Sciences Inc as an investigator-initiated study.

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Results Participant enrolment characteristics

Between July 2014 and December 2015, 257 individuals positive for HIV and anti-HCV antibody were enrolled in CEASE-D (Figure 5-1). Demographic and enrolment characteristics are presented in Table 5-1. The participants were predominantly Caucasian (85%) males (95%; mean age 47 years, SD 9) with well controlled HIV-infection (median CD4 count 587x106/L, IQR 430-800; HIV viral load below the limit of detection, 72%). HCV RNA was detected in 84% (n=215). In those with detectable HCV RNA, the predominant HCV genotypes were 1 (58%; 1a, n=99; 1b, n=12; no subtype, n=14) and 3 (23%). The major modes of HCV acquisition were injecting drug use (IDU) (52%) and sexual exposure in men-who-have-sex- with-men (MSM, 29%). Severe fibrosis or cirrhosis (Metavir F3 or F4) was evident in 19%. Of those individuals who had had transient elastography within the 6 months prior to enrolment, median liver stiffness measurement was 6.2 kPa (IQR 4.9, 8.8 kPa; range 3.0, 65.2 kPa). Thirty-two percent (n=82) had previously received treatment for HCV.

Current combination antiretroviral therapy

Ninety-seven percent of participants were receiving cART (n=249), consisting of combinations of 17 individual antiretroviral agents. For one participant, the current cART regimen was unknown. As expected, participants were receiving a median of 3 antiretroviral drugs (range 2- 6), with 24% receiving 4 (n=59) and 5% receiving ≥5 (n=12) antiretroviral drugs. Thirteen percent (n=32) were receiving antiretroviral drugs from 3 or more classes. Most individuals were receiving a nucleoside/nucleotide reverse transcriptase inhibitor (NRTI/NtRTI) backbone with an integrase inhibitor (II) (37%), non-nucleoside reverse transcriptase inhibitor (NNRTI) (27%) or protease inhibitor (PI) (19%) (Supplementary Material). The three most common cART regimens were tenofovir disoproxil fumarate (TDF) + emtricitabine + efavirenz (12%, n=31), abacavir + lamivudine + dolutegravir (11%, n=27) and TDF + emtricitabine + rilpivirine (10%, n=26). For the cART regimens specific to those with detectable HCV RNA being considered for DAA therapy, see Figure 5-2 and Supplementary Material.

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Table 5-1. Participant enrolment characteristics

On cART Total study Demographic and clinical Detectable Undetectable Missing population characteristics HCV RNA HCV RNA HCV RNA N=257 N=208 N=32 N=9 Age (years), n (%) <30 8 (3) 6 (3) 0 0 30-39 49 (19) 38 (18) 8 (25) 1 (11) 40-49 98 (38) 82 (39) 12 (38) 2 (22) 50-59 76 (30) 58 (28) 10 (31)( 6 (67) ≥60 26 (10) 24 (12) 2 (6) 0 Mean age (SD) 47 (9) 47 (9) 47 (9) 50 (8) Gender, n (%) Male 244 (95) 198 (95) 29 (91) 9 (100) Female 11 (4) 9 (4) 2 (6) 0 Transgender 2 (1) 1 (1) 1 (3) 0 Ethnicity, n (%) Caucasian 219 (85) 178 (86) 26 (81) 8 (89( Asian 19 (7) 15 (7) 2 (6) 1 (11) Hispanic 5 (2) 4 (2) 1 (3) 0 Indian 2 (1) 1 (1) 1 (3) 0 ATSI 4 (2) 4 (2) 0 0 Other/not specified 8 (3) 6 (3) 2 (6) 0 On cART, n (%) 249 (97) a 208 (100) 32 (100) 9 (100) 587 596 553 615 Median CD4 count, x106/L (IQR) (430-800) (436-809) (419-772) (560-836) HIV viral load below limit of 184 (72) 149 (72) 28 (88) 5 (56) detection, n (%) HCV RNA detected, n (%) 215 (84) 208 (100) 0 NA HCV genotype, n (%) b 1 125 (58) 122 (59) NA NA 2 7 (3) 7 (3) NA NA 3 49 (23) 46 (22) NA NA 4 6 (3) 6 (3) NA NA Mixed c 1 (1) 1 (1) NA NA Unknown/missing 27 (13) 26 (13) NA NA Mode of HCV acquisition, n (%) Injection drug use 133 (52) 103 (50) 20 (63) 6 (67) Sexual exposure: MSM 75 (29) 64 (31) 7 (22) 2 (22) Sexual exposure: heterosexual 9 (4) 8 (4) 1 (3) 0 Tattooing 2 (1) 2 (1) 0 0 Transfusion 3 (1) 1 (1) 0 1 (11) Other 3 (1) 2 (1) 2 (6) 0 Unknown/missing 32 (12) 28 (13) 2 (6) 0 Prior HCV therapy, n (%) 82 (32) 58 (28) 19 (59) 5 (56) Metavir fibrosis stage, n (%) ≤F2 164 (64) 137 (66) 20 (63) 2 (22) F3/4 48 (19) 37 (18) 5 (16) 1 (11) Not available 45 (18) 29 (14) 7 (22) 6 (67) a cART regimen unknown for one individual b HCV genotype distribution in those with detectable HCV RNA c Mixed HCV genotype – GT 1a and 3 Abbreviations: ATSI, Aboriginal/Torres Strait islander; cART, combination antiretroviral therapy

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Participants on Antiretroviral SOF SOF/LDV SOF/VEL DCV PrOD PrO GZR/EBR SIM RBV antiretroviral n (%) NRTI/NtRTI Lamivudine 62 (30) Abacavir 56 (27) Emtricitabine 135 (65) Monitor renal

Tenofovir (TDF) 140 (67) function NNRTI ↑DCV 90mg

Nevirapine 9 (4) daily Monitor renal ↑DCV 90mg

Efavirenz 33 (16) function daily ↑DCV 90mg

Etravirine 8 (4) daily Potential QTc Potential QTc

Rilpivirine 24 (12) prolongation prolongation Protease inhibitor Monitor

Atazanavir 2 (1) bilirubin ↓DCV 30mg Monitor

Atazanavir/r 21 (10) daily bilirubin Darunavir/r 23 (11) Lopinavir/r 12 (6) ↓DCV 30mg

Saquinavir 1 (0.5) daily Integrase or entry inhibitor Raltegravir 41 (20) Dolutegravir 45 (22) Monitor renal ↓DCV 30mg

Elvitegravir/c 11 (5) function daily Maraviroc 5 (2)

No clinically significant Potential significant Co-administration Figure legend No data interaction interaction contraindicated

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Figure 5-2. Concomitant use of antiretroviral drugs and approved interferon-free DAA regimens in the CEASE-D cohort

Antiretroviral drugs prescribed for participants with detectable HCV RNA, regardless of HCV genotype.

Antiretroviral agents involved in drug–drug interactions (DDIs) and suggested actions per DAA regimen. Colour code is as follows: green, category 1, no significant DDI; yellow, category 2, potentially significant DDI possible; and red, category 3, co-administration either not recommended or contra- indicated. The clinical significance of the drug interaction is based on individual DAA prescribing information and www.hep-druginteractions.org.

Abbreviations: c, cobicistat; D, dasabuvir; DCV, daclatasvir; GZR/EBR, grazoprevir/elbasvir; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI/NtRTI, nucleoside/nucleotide reverse transcriptase inhibitor; PrO, paritaprevir/ritonavir/ombitasvir fixed dose combination; r, ritonavir; RBV, ribavirin; SIM, simeprevir; SOF, sofosbuvir; SOF/LDV, sofosbuvir/ledipasvir fixed dose combination; SOF/VEL, sofosbuvir/velpatasvir fixed dose combination;

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Drug-drug interactions between DAAs and cART

Prescribed antiretroviral agents in those with detectable HCV RNA and their potential for DDIs with interferon-free DAAs in this cohort are displayed in Table 5-2 and Figure 5-2. The risk of a clinically significant DDI with currently prescribed cART varied markedly between DAA regimens.

In participants on cART with HCV genotype 1 and 4 and detectable HCV RNA (n=128) (Supplementary Figure 5-1), category 1(no clinically significant interaction) DDIs were expected in 29% with sofosbuvir/ledipasvir, 59% with sofosbuvir plus daclatasvir, 73% with sofosbuvir/velpatasvir, 36% with PrO +/- D (+/-ribavirin), 49% with grazoprevir/elbasvir and 49% with sofosbuvir plus simeprevir. Category 2 DDIs were expected in 71% with sofosbuvir/ledipasvir, 41% with sofosbuvir plus daclatasvir, and 28% with PrO +/- D (+/-ribavirin). No category 2 DDIs were expected with sofosbuvir/velpatasvir, grazoprevir/elbasvir and sofosbuvir plus simeprevir. Specifically, category 1 and 2 DDIs were expected in 35% and 30%, respectively, with PrOD in HCV GT 1 and in 50% and 0%, respectively, with PrO in HCV GT 4. In the case of sofosbuvir plus daclatasvir, all category 2 DDIs involved DAA dose adjustment; an increase in daclatasvir dose to 90mg daily would be required in 23% (n=30) due to an interaction with a NNRTI (efavirenz, n=22; etravirine, n=7; nevirapine, n=3) and a reduction in daclatasvir dose to 30mg daily would be required in 16% (n=21) due to an interaction with a pharmacokinetic booster (atazanavir/ritonavir, n=12; saquinavir/ritonavir, n=1; elvitegravir/cobicistat, n=8). Category 2 DDIs that would require minor antiretroviral adjustment (ritonavir-boosted atazanavir, n=11 or darunavir, n=7) were noted in 14% with PrO +/- D. Category 3 DDIs (contra-indicated or not recommended for co-administration) were noted in 36% with PrO +/- D, 51% with grazoprevir/elbasvir, 51% with sofosbuvir plus simeprevir, and 17% with sofosbuvir/velpatasvir. No category 3 DDIs were expected with sofosbuvir/ledipasvir and sofosbuvir plus daclatasvir. The antiretroviral drug classes associated with category 3 DDIs were predominantly the NNRTIs and HIV PIs (PrOD, n=62 – NNRTI 77%, PI 29%, II with cobicistat 13%; grazoprevir/elbasvir, n=65 – NNRTI 49%, PI 46%, II with cobicistat 12%; sofosbuvir plus simeprevir, n=65 – NNRTI 49%, PI 46%, II with cobicistat 12%; sofosbuvir/velpatasvir, n=22 – NNRTI 100%). No data is available for the potential DDIs between sofosbuvir/velpatasvir and nevirapine, etravirine and maraviroc (category 4 DDI, 9%). Given the known interaction with efavirenz, it would be expected that co-administration of sofosbuvir/velpatasvir and nevirapine or etravirine would be contraindicated. The current cART regimens were suitable for co-administration with sofosbuvir/ledipasvir and sofosbuvir plus daclatasvir in 100% and 100%, respectively. However, DDIs impacted on the suitability for co-administration of the current cART regimens and sofosbuvir/velpatasvir (73%), PrO +/- D (64%), grazoprevir/elbasvir (49%) and sofosbuvir plus simeprevir (49%).

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Table 5-2. Suitability of current cART regimen for co-administration with DAA regimen by HCV genotype

HCV GT HCV GT 1 and 4 HCV GT 2 and 3 indeterminate/unknown n=128 n=531 DDI category, n (%) n=26 PrO+/-D SOF + SOF + SOF + SOF + SOF/LDV GZR/EBR SOF/VEL SOF + RBV SOF/VEL SOF/VEL +/- RBV SIM DCV DCV DCV Category 1: No significant DDI 37 (29) 46 (36) 63 (49) 63 (49) 75 (59) 94 (73) 47 (89) 36 (68) 43 (81) 12 (46) 17 (65)

Category 2: Potentially significant DDI 91 (71) 36 (28) 0 0 53 (41) 0 6 (11) 17 (32) 0 14 (54) 0

Adjust DAA 53 (41) 17 (32) 14 (54)

Additional monitoring 91 (71) 18 (14) 6 (11) Adjust cART dose or timing of administration 18 (14) Category 3: Not recommended or contra-indicated 0 46 (36) 65 (51) 65 (51) 0 22 (17) 7 (13) 5 (19) Category 4: No data 0 0 0 0 0 12 (9)2 3 (6)2 4 (15)2

Suitable for co-administration 128 (100) 82 (64) 63 (49) 63 (49) 128 (100) 94 (73) 56 (100) 56 (100) 43 (81) 26 (100) 17 (65)

Antiretroviral class associated with category 3

DDI3 NNRTI 31 (67) 31 (48) 31 (48) 22 (100) 7 (100) 5 (100) PI 12 (26) 31 (48) 31 (48) Integrase inhibitor with cobicistat 8 (17) 8 (12) 8 (12)

1 Includes one participant with mixed infection (GT 1a/3a) 2 No data available for co-administration of maraviroc, nevirapine or etravirine with SOF/VEL; given the interaction with efavirenz, category 3 DDI expected with nevirapine and etravirine. 3 Individuals may be prescribed more than one antiretroviral class resulting in a category 3 DDI (not recommended or contra-indicated)

Abbreviations: DCV, daclatasvir; DDI, drug-drug interaction; GT, genotype; GZR/EBR, grazoprevir/elbasvir; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI/NtRTI, nucleoside/nucleotide reverse transcriptase inhibitor; PrO +/- D +/- RBV: Ombitasvir/paritaprevir/ritonavir fixed dose combination with or without dasabuvir (without ribavirin in genotype 1b); SOF, sofosbuvir; SOF/LDV, sofosbuvir/ledipasvir fixed dose combination; SOF/VEL, sofosbuvir/velpatasvir fixed dose combination

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In participants on cART with HCV genotype 2 and 3 and detectable HCV RNA (genotype 2, n=7; genotype 3, n=46, including 1 mixed genotype 1a/3a infection), category 1 DDIs were expected in 89% with sofosbuvir plus ribavirin, 68% with sofosbuvir plus daclatasvir and 81% with sofosbuvir/velpatasvir (Supplementary Figure 5-2). Category 2 DDIs were expected in 11% with sofosbuvir plus ribavirin, 32% with sofosbuvir plus daclatasvir and 0 with sofosbuvir/velpatasvir. All category 2 DDIs related to sofosbuvir and daclatasvir involved dose adjustment of daclatasvir (n=17; elvitegravir/cobicistat, n=2; efavirenz, n=7; nevirapine, n=3; ritonavir-boosted atazanavir, n=5). No category 3 DDIs were noted with sofosbuvir plus ribavirin and sofosbuvir plus daclatasvir. However, category 3 and 4 DDIs were noted in 13% and 6%, respectively, with sofosbuvir/velpatasvir (with all category 3 DDIs related to efavirenz and all category 4 DDIs related to nevirapine). Current cART regimens were suitable for co- administration with sofosbuvir plus ribavirin and sofosbuvir plus daclatasvir in 100%, with no antiretroviral alterations required. With sofosbuvir/velpatasvir, current cART regimens were suitable for co-administration in 81%.

In participants on cART with HCV GT indeterminate or unknown (n=26), two pan-genotypic regimens were assessed. Category 1 DDIs were expected in 46% with sofosbuvir plus daclatasvir and 65% with sofosbuvir/velpatasvir. Category 2 DDIs were expected in 54% with sofosbuvir plus daclatasvir and 0 with sofosbuvir/velpatasvir. No category 3 DDIs were noted with sofosbuvir plus daclatasvir. Category 3 and 4 DDIs were noted in 19% and 15%, respectively, with sofosbuvir/velpatasvir (with all category 3 DDIs related to efavirenz and all category 4 DDIs related to nevirapine or etravirine). Current cART regimens were suitable for co-administration with sofosbuvir plus daclatasvir in 100% and sofosbuvir/velpatasvir in 65%.

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Discussion The availability of highly effective, well tolerated interferon-free DAA regimens for HCV should diminish barriers to therapy in HIV/HCV co-infection. However, treatment of HIV/HCV co-infected individuals will require an awareness of the potential DDIs between specific DAAs and HIV antiretroviral agents by both prescribers and clinical pharmacists in order to prevent morbidity and ensure treatment efficacy. In this real-world cohort of HIV/HCV co-infected individuals, there was significant potential for DDIs between currently prescribed cART and approved interferon-free DAA regimens. Most participants were receiving cART regimens which were suitable for co-administration with sofosbuvir and a first generation NS5A inhibitor. However, based on current prescribing information, the DAA regimens including HCV NS3/4a protease inhibitors were not appropriate for co-administration in more than half of study participants. Additionally, HIV/HCV co-infected individuals need to have achieved HIV RNA suppression prior to initiation of PrO+/-D, as the low-dose ritonavir required to boost paritaprevir may select for HIV PI resistance; 28% of the CEASE-D cohort did not demonstrate HIV RNA suppression.

Representative of the broader HIV/HCV-infected population in many countries, most individuals in this cohort had HCV genotype 1 infection, which has significant implications for choice of DAA regimen. Only 16% (n=20) of those with HCV genotype 1 or 4 were receiving cART which demonstrated no clinically significant DDIs with all of the assessed interferon-free DAA regimens; those without any significant DDIs were all prescribed two NRTIs (abacavir/lamivudine) and an integrase inhibitor (dolutegravir or raltegravir).

While antiretroviral switches may be performed to allow co-administration with specific DAAs (347), as we have demonstrated in the CEASE-D cohort, most HIV-positive individuals on cART, even those receiving complex regimens with agents from 3 or more classes, should be able to receive a suitable interferon-free HCV DAA regimen (in line with current international guidelines(91)) without altering their current antiretroviral regimen. This is important to note in the context of current limitations or restrictions placed upon DAA access in many countries, largely mediated by payers. The flexibility to individualise therapy and prescribe an appropriate DAA regimen is essential to maximise safety and efficacy. However, if required, changes in the antiretroviral regimen should be undertaken in collaboration with a HIV physician(91). As international cART guidelines change, regimens favouring integrase inhibitor use are anticipated, which should reduce the proportion with significant DDIs(355).

DDI management presents increasing challenges as the number of drugs prescribed increases per individual; in this cohort, for those on cART, 98% took 3 or more drugs, irrespective of other concomitant medications and prior to DAA prescription. To date, most HIV/HCV co-

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infected individuals have been treated in specialist centres. However, as DAA prescription becomes increasingly commonplace outside of these settings, recognition of relevant DDI with cART remains important for optimal management of co-infected patients.

Primarily, two scenarios need to be considered and avoided; an increase in plasma drug levels, potentially leading to adverse events and a reduction in plasma drug levels, potentially resulting in loss of efficacy. Considering commonly prescribed antiretroviral drugs within the CEASE-D cohort, particular DDIs and potentially significant clinical events are notable. In line with international guidelines, tenofovir-containing cART regimens were commonly prescribed in this cohort (65%). An increase in tenofovir concentrations when TDF is co-administered with sofosbuvir/ledipasvir and efavirenz, rilpivirine or a boosted-protease or integrase inhibitor has raised concerns regarding nephrotoxicity (351). However, data from clinical trials and real- world cohorts provides some reassurance (229, 244, 348). In the Phase III ION-4 trial, only 1% of participants were noted to have an increase in baseline serum creatinine ≥0.4 mg/dl (≥35 μmol/L) while on treatment(229). Additionally, recent FDA approval of tenofovir alafenamide (TAF) provides a potentially safer alternative for co-administration if concerns regarding renal toxicity persist(353). HIV protease inhibitors were prescribed in 29%, with implications for daclatasvir and HCV NS3/4a protease inhibitors. Concomitant use of elbasvir/grazoprevir with HIV protease inhibitors is contra-indicated due to organic anion-transporting polypeptide (OATP) 1B inhibition and resultant marked increase in grazoprevir AUC and potential for ALT elevation(352). A reduction in DAA drug level may impact on sustained virological response and selection of resistance-associated variants (356). Efavirenz (prescribed in 15%), an inducer of CYP3A, markedly reduces grazoprevir/elbasvir (352), PrO (211), velpatasvir (354) and daclatasvir serum concentrations (191). As such, efavirenz is contra-indicated with grazoprevir/elbasvir, PrO and sofosbuvir/velpatasvir and an increase in daclatasvir dose is necessary if co-administered with efavirenz, etravirine or nevirapine; this latter DDI could impact 25% of the CEASE-D cohort. A reduction in antiretroviral drug level may lead to HIV virological failure. Darunavir serum trough concentrations are reduced by 50% when co- administered with PrO, so caution should be exercised in individuals with a history of HIV protease inhibitor resistance (357).

The main limitation of this study is that cohort enrolment is currently restricted to five treatment centres in Sydney, Australia, which may limit generalisability. However, given that antiretroviral use in CEASE-D is similar to that in the overall Australian HIV Observational Database, our results are likely to be applicable to the broader HIV/HCV population in Australia and representative of co-infected populations in many high income settings. Given the extensive use of TDF + emtricitabine + efavirenz in HIV-positive populations in low and middle income countries, the choice of DAA regimen in those with HIV/HCV co-infection will

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be impacted by potential DDIs. Sofosbuvir plus daclatasvir would be suitable in this setting given its pan-genotypic activity and the ability to dose adjust daclatasvir. Other limitations are the lack of data to determine what proportion of individuals could safely switch antiretroviral drugs for the duration of their HCV treatment and the inability to assess other co-morbidities and competing polypharmacy in this cohort.

While offering greater efficacy, tolerability and simplicity than interferon-containing regimens, DDIs will impact on DAA prescribing in HIV/HCV co-infection. The combinations of sofosbuvir plus an NS5A inhibitor and sofosbuvir plus ribavirin appear to be suitable for co- administration with commonly used antiretroviral agents, making these DAA regimens appealing for use in HIV/HCV co-infection. However, the use of an HCV NS3/4a protease inhibitor-containing DAA regimen poses more challenges. The involvement of clinical pharmacists in assessing DDI risk prior to commencing DAA therapy may be warranted. Evaluation of potential DDIs is required to prevent adverse events or treatment failure.

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Supplementary Material

Tables Supplementary Table 5-1. Summary of major metabolic pathways of approved DAAs which may be implicated in drug-drug interactions

Drug, by class Substrate of Inhibitor of Transported by

NS5B polymerase inhibitors CatA P-gp Sofosbuvir CES1 P-gp (gut) BCRP Hint1 CYP2C8 UGT1A1 P-gp Dasabuvir CYP3A OATP1B1 BCRP NS3/4A protease inhibitors CYP3A4 P-gp (gut) P-gp CYP3A4 Paritaprevir/r UGT1A1 BCRP CYP3A5 OATP1B1/B3 OATP1B1/B3 BCRP CYP2C8 P-gp Grazoprevir CYP3A4 CYP3A4 OATP1B1/B3 UGT1A1 CYP1A2 P-gp CYP3A4 (gut) BCRP Simeprevir CYP3A OATP1B1/B3 OATP1B1/B3 MRP2 OATP2B1 NS5A inhibitors P-gp Daclatasvir CYP3A4 OATP1B1/B3 P-gp BCRP P-gp (gut) Slow oxidative P-gp Ledipasvir BCRP metabolism BCRP OATP1B1/B3 Amide hydrolysis, then CYP2C8 P-gp Ombitasvir oxidative metabolism UGT1A1 BCRP P-gp Elbasvir CYP3A4 - CYP3A4 OATP P-gp CYP2B6 P-gp BCRP Velpatasvir CYP2C8 BCRP OATP1B1/B3 CYP3A4 OATP1B1/B3 OATP2B1

Abbreviations: BCRP, breast cancer resistance protein; CatA, human cathepsin A; CES1, carboxylesterase 1; Hint1, histidine triad nucleotide-binding protein 1; OATP, organic anion-transporting polypeptide; P-gp, P-glycoprotein; UGT, uridine diphosphate-glucuronosyltransferase

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Supplementary Table 5-2. Permitted HIV antiretroviral agents in phase II and III clinical trials of interferon-free DAAs in HIV/HCV co-infection

DAA HCV Treatment SVR 12 Ref Trial acronym Permitted HIV antiretroviral agents regimen GT duration (%) 2 NRTIs (FTC/TDF) plus: • Efavirenz (n=16) (244) ERADICATE SOF/LDV 1 12 weeks 98 • Raltegravir (n=14) • Rilpivirine (n=11) 2 NRTIs (FTC/TDF) plus: • Efavirenz (n=160) (229) ION-4 SOF/LDV 1 12 weeks 96 • Raltegravir (n=146) • Rilpivirine (n=29) 2 NRTIs plus: • PI (darunavir/r, n=51; atazanavir/r, n=36; lopinavir/r, n=12) SOF + 8 weeks 76 (237) ALLY-2 1-4 • NNRTI (efavirenz, n=34; nevirapine, DCV 12 weeks 97 n=9; rilpivirine, n=7) • Integrase inhibitor (raltegravir, n=40; dolutegravir; n=8) 2 NRTIs (FTC, TDF or lamivudine) plus: PrOD+/- 12 weeks 94 (238) TURQUOISE-I 1 • Atazanavir (n=28) RBV 24 weeks 91 • Raltegravir (n=35) 87 (without GZR/EBR RBV) 2 NRTIs (FTC/TDF) plus: (245) C-WORTHY 1 12 weeks +/- RBV 97 (with • Raltegravir (n=59) RBV) 2 NRTIs (TDF or abacavir + FTC or lamivudine) plus: (239) C-EDGE GZR/EBR 1, 4, 6 12 weeks 96 • Raltegravir (n=113) • Dolutegravir (n=59) • Rilpivirine (n=38) 2 NRTIs (TDF or abacavir + FTC or lamivudine) plus: • PI (darunavir, lopinavir or atazanavir, n=50) (240) ASTRAL 5 SOF/VEL 1-4 12 weeks 95 • NNRTI (rilpivirine, n=13) • Integrase inhibitor (dolutegravir or elvitegravir, n=34) • PI +/- NNRTI +/- integrase inhibitor (n=7) G2 88 2 NRTIs (FTC/TDF ) plus: 12 weeks G3 67 • PI (atazanavir/r, n=39; darunavir/r, n=34) SOF + (242) PHOTON-1 1-3 G1 76 • NNRTI (efavirenz, n=78; rilpivirine, RBV 24 weeks G2 92 n=14) G3 94 • Integrase inhibitor (raltegravir, n=36) • Other (n=10) 2 NRTIs (FTC/TDF) plus: 12 weeks G2 88 • PI (atazanavir/r, n=44; darunavir/r, n=56) (243) PHOTON-2 SOF+RBV 1-4 G1 85 • NNRTI (efavirenz, n=64; rilpivirine, 24 weeks G3 89 n=12) G4 84 • Integrase inhibitor (raltegravir, n=61) • Other (n=27) Abbreviations: DCV, daclatasvir; FDC, fixed dose combination; FTC, emtricitabine; GZR/EBR, grazoprevir/elbasvir; NRTI/NtRTI, nucleoside/nucleotide reverse transcriptase inhibitor; NNRTI, non- nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; PrOD, Paritaprevir/ritonavir/ombitasvir fixed dose combination with dasabuvir; RBV, ribavirin; SOF, sofosbuvir; SOF/LDV, sofosbuvir/ledipasvir fixed dose combination; SOF/VEL, sofosbuvir/velpatasvir; TDF, tenofovir disoproxil fumarate

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Supplementary Table 5-3. Proportion of individuals receiving specific antiretroviral agents Total cohort Detectable HCV RNA Antiretroviral agent by class (n=249)* (n=208)* NRTI/NtRTI, n (%) 239 (96) 201 (97) Abacavir 68 (27) 56 (27) Emtricitabine 157 (63) 135 (65) Lamivudine 74 (30) 62 (30) Tenofovir (TDF) 163 (65) 140 (67) NNRTI, n (%) 91 (37) 74 (36) Efavirenz 37 (15) 33 (16) Etravirine 11 (4) 8 (4) Nevirapine 15 (6) 9 (4) Rilpivirine 28 (11) 24 (12) Protease inhibitor, n (%) 73 (29) 58 (28) Atazanavir 3 (1) 2 (1) Atazanavir/ritonavir 27 (11) 21(10) Darunavir/ritonavir 29 (12) 23 (11) Lopinavir/ritonavir 14 (6) 12 (6) Saquinavir/ritonavir 1 (0) 1 (0) Integrase inhibitor, n (%) 119 (48) 97 (47) Dolutegravir 54 (22) 45 (22) Elvitegravir/cobicistat 16 (6) 11 (5) Raltegravir 49 (20) 41 (20) Entry inhibitor, n (%) 7 (3) 5 (2) Maraviroc 7 (3) 5 (2) Once daily FDC, n (%) 79 (32) 68 (33) Abacavir/lamivudine/dolutegravir 12 (5) 11 (5) TDF/emtricitabine/efavirenz 30 (12) 29 (14) TDF/emtricitabine/elvitegravir/cobicistat 16 (6) 11 (5) TDF/emtricitabine/rilpivirine 21 (8) 17 (8) NRTI/NtRTI + II 91 (37) 77 (37) NRTI/NtRTI + NNRTI 68 (27) 58 (28) NRTI/NtRTI + PI 47 (19) 41 (20) II + PI +/- NRTI 12 (5) 9 (4) NNRTI + II +/- NRTI 9 (4) 7 (3) NNRTI + PI +/- NRTI 5 (2) 3 (1) NNRTI + II + PI +/- NRTI or Entry inhibitor 7 (3) 4 (2) NRTI/NtRTI only 3 (1) 3 (1) NRTI/NtRTI + Entry inhibitor 2 (1) 2 (1) NRTI/NtRTI + NNRTI + Entry inhibitor 2 (1) 2 (1) NRTI/NtRTI + PI + Entry inhibitor 1 (0) 0 PI only 1 (0) 1 (0) Unknown 1 (0) 1 (0) *cART regimen unknown for 1 individual

Abbreviations: FDC, fixed dose combination; II, integrase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI/NtRTI, nucleoside/nucleotide reverse transcriptase inhibitor; PI, protease inhibitor; TDF, tenofovir disoproxil fumarate

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Figures

100%

80%

60%

40%

20%

0% SOF/LDV PrO+/-D GZR/EBR SOF+SIM SOF+DCV SOF/VEL

Category 4: No data Category 3: Co-administration not recommended or contra-indicated Category 2: Potentially significant DDI Category 1: No significant DDI

Supplementary Figure 5-1. Proportion of participants with significant drug–drug interactions between their cART and DAA regimens - HCV genotype 1 and genotype 4

Participants were categorized according to the drug with the highest DDI risk in their cART regimen. Category 1 and 2 DDIs were considered suitable for co-administration with the DAA regimen.

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100%

80%

60%

40%

20%

0% SOF+RBV SOF+DCV SOF/VEL Category 4: No data Category 3: Co-administration not recommended or contra-indicated Category 2: Potentially significant DDI Category 1: No significant DDI

Supplementary Figure 5-2. Proportion of participants with significant drug–drug interactions between their cART and approved DAA regimens - HCV genotype 2 and genotype 3

Participants were categorized according to the drug with the highest DDI risk in their cART regimen. Category 1 and 2 DDIs were considered suitable for co-administration with the DAA regimen.

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Chapter 6 Integrated Discussion The broad aim of the research described in this thesis was to evaluate novel therapeutic strategies in recent HCV infection (estimated infection duration <18 months) following the development of HCV direct-acting antiviral (DAA) therapy. There are six specific aims and hypotheses, which include examining the feasibility, efficacy, and safety of response-guided interferon-containing and ultra-short duration interferon-free therapy. In this chapter, the key findings of the research are summarised with respect to the six specific aims and hypotheses. The implications of the key findings, directions for future research and thesis strengths and limitations are also discussed.

Key Findings

Aim 1: To evaluate the efficacy and safety of response-guided interferon-containing therapy in individuals with recent HCV infection Hypothesis: Response-guided therapy will be effective and safe in most individuals with recent HCV infection.

This aim is addressed in Chapter Two.

The efficacy and safety of response-guided interferon-based therapy for recent HCV infection (estimated duration of infection ≤18 months) was evaluated in two prospective, multicentre, open-label trials: the Australian Trial in Acute Hepatitis C II (ATAHC II), in which treated participants received pegylated-interferon alfa-2a (PEG-IFN) with or without ribavirin, and the Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C trial (DARE-C I), in which participants received PEG-IFN, ribavirin and telaprevir. Treatment duration was dependent on time to first HCV RNA below the level of detection (ATAHC II: 8, 16, 24 or 48 weeks; DARE-C I: 8, 12 or 24 weeks). The primary efficacy endpoint was SVR12 in the ITT population.

ATAHC II demonstrated that recent HCV infection can be effectively treated with response guided PEG-IFN and ribavirin. The majority of treated participants (56%) were able to receive short duration (8-16 weeks) therapy, with the overall SVR (SVR12 71% [37/52]) similar to that observed with previously recommended 24 week regimens (133). On logistic regression analysis, SVR12 was associated with a rapid virological response (OR 10.80; 95% CI 2.51,

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46.43; p=0.001). The safety profile was consistent with the known side effects of PEG-IFN alfa-2a and ribavirin. At least one clinical adverse event was reported by 51 participants (98%) with most adverse events of mild (75%) or moderate (24%) severity. PEG-IFN and ribavirin dose modification were required for toxicity in 6% (neutropenia, n=3) and 2% (anaemia, n=1), respectively. Three serious adverse events were reported, of which two were possibly related to study drug administration. No decompensated liver disease or death occurred.

In the DARE-C I study, response-guided therapy with PEG-IFN, ribavirin and telaprevir was effective in the majority (SVR12 71%, [10/14]), although similar to that observed with PEG- IFN and ribavirin alone. Multiple adverse events were documented in all participants with the most common being fatigue (73%) and rash (50%). Three serious adverse events were reported; all were deemed to be unrelated to study drug administration. Adverse events requiring medical intervention, treatment cessation or dose modification occurred in 36% (n=5), with dose reduction of PEG-IFN and ribavirin in one (7%) and three (21%) individuals, respectively. The addition of telaprevir was associated with excess haematological toxicity. Mean decrease in haemoglobin at end of treatment was 33 g/L (SD 18) in participants receiving PEG-IFN, ribavirin and telaprevir as compared with 20 g/L (SD 15) in participants receiving PEG-IFN and ribavirin (p=0.007). Anaemia (haemoglobin less than 100 g/L) developed on treatment in 5 participants (36%) receiving PEG-IFN, ribavirin and telaprevir as compared with 3 (6%) receiving PEG-IFN and ribavirin (p=0.008). Despite the short treatment duration, the side- effect profile, drug-drug interactions and treatment complexity seen with this regimen indicate that the addition of telaprevir offers no significant benefit.

While response-guided interferon-based therapy had reasonable efficacy in acute and recent HCV infection, the side effect profile would limit implementation. In the absence of interferon- free therapy, a response-guided strategy using PEG-IFN and ribavirin in recent HCV infection could be considered for motivated individuals wishing to trial therapy, with treatment cessation at week four if HCV RNA remains detectable.

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Aim 2: To evaluate the feasibility, efficacy and safety of ultra-short duration interferon- free therapy in individuals with recent HCV infection Hypothesis: Short duration interferon-free therapy will be safe and effective in most individuals with recent HCV infection.

This aim is addressed in Chapter Three.

The feasibility, efficacy and safety of ultra-short duration interferon-free therapy for recent HCV infection were assessed in the Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C II trial (DARE-C II). Participants with recent HCV infection (estimated duration of infection <12 months) received sofosbuvir 400mg daily and weight-based ribavirin for six weeks. The primary efficacy endpoint was SVR12 in the ITT population.

Nineteen participants commenced sofosbuvir and ribavirin (89% male, 74% HIV, 68% genotype 1a). Twenty-one percent had a symptomatic seroconversion illness (n=4, including 2 with jaundice). At baseline, median HCV RNA was 5.4 log10 IU/mL (IQR 4.4-6.8) and median estimated duration of infection was 37 weeks (IQR 27-41). At end-of-treatment, HCV RNA was non-quantifiable in 89% (n=17). SVR4 and SVR12 were 42% (n=8) and 32% (n=6), respectively. Treatment failure was due to non-response (n=2), post-treatment relapse (n=9), reinfection (n=1) and loss to follow up (n=1). The regimen was well tolerated with minimal haematological toxicity. SVR12 was related to baseline HCV RNA (≤6 log10 IU/mL, p=0.018) and early on-treatment viral kinetics (HCV RNA below the level of quantitation at week 1, p=0.003).

While six weeks of sofosbuvir and ribavirin was safe and well tolerated, efficacy was poor. This study conclusively demonstrated that while on-treatment viral suppression was achieved in the majority, a high proportion of participants demonstrated post-treatment relapse. Baseline

HCV RNA ≤6 log10 IU/mL and subsequent rapid viral suppression (HCV RNA below the LLoQ [TND or TDnq] at week 1 and HCV RNA TND at week 2) were associated with SVR12, supporting further research with more potent DAA regimens in recent HCV infection. In the era of DAA therapy, it is uncertain whether acute or recent HCV infection will continue to offer a unique therapeutic advantage, as compared with chronic HCV infection.

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Aim 3: To evaluate the efficacy of response-guided interferon-containing and short duration interferon-free therapy in HCV mono-infection as compared with HIV/HCV co- infection Hypothesis: Short duration and/or response-guided therapy will be effective in most individuals with recent HCV infection, regardless of HIV serostatus.

This aim is addressed in Chapter Two and Three.

The efficacy of response-guided interferon-containing therapy was not compromised by HIV co-infection. Fifty-two participants (71% HIV-positive) received treatment in ATAHC II and 14 (79% HIV-positive) in DARE-C I. In ATAHC II, overall SVR12 by ITT was 71% (37/52; 95% CI 57%, 83%), with no difference by HIV serostatus (HCV mono-infection, SVR12 73% [11/15]; HIV/HCV co-infection, SVR12 70% [26/37]; p=0.825). In DARE-C I, overall SVR12 by ITT was 71% (10/14; 95% CI 42%, 92%); acknowledging the small treated population, there was no significant difference by HIV serostatus (HCV mono-infection, SVR12 66% [2/3]; HIV/HCV co-infection, SVR12 73% [8/11]; p=1.000).

Similarly, HIV co-infection did not significantly impact the efficacy of ultra-short duration sofosbuvir and ribavirin In DARE-C II, overall SVR12 ITT was 32% (6/19; 95% CI 13%, 57%). By ITT analysis, SVR4 ITT and SVR12 ITT were 36% (5/14) and 21% (3/14), respectively, in those with HIV co-infection, as compared with 60% (3/5) and 60% (3/5), respectively, in those with HCV mono-infection (SVR4, p=0.603; SVR12, p=0.262). However, given the small sample size, the impact of HIV co-infection on the efficacy of short duration DAA therapy in recent HCV infection requires further investigation.

Aim 4: To evaluate treatment adherence in individuals receiving response-guided interferon-containing and short duration interferon-free therapy with recent HCV infection Hypothesis: Short duration therapy will enhance treatment adherence.

This aim is addressed in Chapter Two and Three.

Within ATAHC II, adherence to therapy was high with PEG-IFN 80/80 (≥80% of doses for ≥80% of treatment period) and 100/100 (100% of doses for 100% of treatment period) adherence 100% and 98%, respectively (mean on-treatment PEG-IFN adherence 99.96% [SD 0.28%]) and ribavirin 80/80 and 100/100 adherence 94% and 67%, respectively (mean on-

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treatment ribavirin adherence 95.25% [SD 18.65%]). HIV/HCV co-infected participants were more likely to be ribavirin adherent than HCV mono-infected participants (ribavirin 80/80 100% vs 79%, p=0.004; ribavirin 100/100 76% vs 43%, p=0.045). PEG-IFN 80/80 adherence was better with response guided therapy in ATAHC II as compared with the standard 24 week duration in ATAHC I (100% vs 82%; p=0.001).

Similarly in DARE-C I, adherence was high with PEG-IFN 100/100 adherence 100% and ribavirin 80/80 and 100/100 adherence 100% and 50%, respectively (mean on-treatment ribavirin adherence 98.9% [SD 1.6]). Telaprevir 80/80 and 100/100 adherence were 100% and 64% (mean on-treatment telaprevir adherence 98.9% [SD 1.7]).

Of the 19 participants who commenced treatment in DARE-C II, 18 (95%) completed the scheduled six week treatment course. Adherence to therapy was high, by pill count and self- reported questionnaire. Sofosbuvir 90/90 (≥90% of doses for ≥90% of treatment period) and 100/100 adherence were 89% and 79%, respectively, with mean on-treatment sofosbuvir adherence 96.24% (SD 13.62%). Ribavirin 90/90 and 100/100 adherence were 89% and 84%, respectively, with mean on-treatment ribavirin adherence 98.18% (SD 4.73%). Consistent with self-reported adherence, when sampled, all participants had detectable ribavirin plasma concentrations on treatment.

Combined, these trials confirm the feasibility of short duration therapy in those with recent HCV infection, regardless of treatment regimen, with excellent adherence in this historically difficult-to-treat population.

Aim 5: To calculate the incidence of HCV reinfection among individuals treated for recent HCV infection. Hypothesis: HCV reinfection following treatment-induced clearance will occur in the context of ongoing risk behaviour

This aim is addressed in Chapter Four.

Individuals with recent HCV infection (estimated duration of infection <18 months) who received treatment in four prospective open-label studies (ATAHC I, ATAHC II, DARE-C I and DARE-C II) in Australia and New Zealand were assessed for HCV reinfection (133, 332, 337). Between 2004 and 2015, 196 participants with recent HCV infection were included in the intention-to-treat population with an end of treatment response (ETR) in 77% (n=151). Six

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participants (4%) were lost to follow up (LTFU) after ETR. Viral recurrence following ETR was seen in 35 (23%), confirmed by sequencing as relapse in 25 (17%) and reinfection in 10 (6%; eight confirmed, two possible). Excluding those with relapse and LTFU, the cohort at risk for reinfection consisted of 120 treated participants. Total follow-up time post treatment was 141 py (median 1.22 py, range 0.19, 2.53). Total follow-up time at-risk for reinfection (censured at estimated date of reinfection) was 135 py (median 1.08 years, range 0.17, 2.53).

In the cohort at risk for reinfection (median age 36 years [IQR 29-46], male 83%, HIV co- infection 53%, injection drug use ever 69%), high levels of risk behaviour facilitating HCV transmission were reported, including 38% reporting injection drug use at end of and/or post treatment. All cases of reinfection (n=10) occurred in men, the majority of whom were HIV co- infected (70%) and reported injection drug use at end of and/or post treatment (80%). The overall incidence of HCV reinfection was 7.4 per 100 py (95% CI 4.0, 13.8). HCV reinfection incidence was significantly higher amongst participants who reported injection drug use at end of and/or post treatment (15.5 per 100 py, 95% CI 7.8, 31.1) as compared with those who did not inject drugs during follow up (2.6 per 100 py, 95% CI 0.6, 10.3) (p=0.023). In adjusted exact Poisson regression analysis, reinfection was associated with older age (aIRR 5.42, 95% CI 1.06, 52.93, p=0.040) and injection drug use during follow up (aIRR 7.86, 95%CI 1.54, 76.79, p=0.008). Similarly, among HIV-positive MSM, injection drug use at end of and/or post treatment was the only factor associated with HCV reinfection in this sub-group (aIRR 8.19, 95%CI 1.34, 85.99, p=0.019; adjusted for age). Amongst PWID (including HIV-positive MSM), HCV reinfection was associated with use of unsterile needles and syringes during follow up (aIRR 16.70, 95%CI 3.20, 108.88, p<0.001; adjusted for age).

The significant risk for HCV reinfection following treatment in individuals with ongoing high risk behaviour facilitating transmission emphasises the need for post-treatment surveillance, harm reduction strategies and education (270, 311).

Aim 6: To assess the clinical significance of drug-drug interactions between HCV DAAs and HIV antiretroviral therapy. Hypothesis: Drug-drug interactions will occur and will impact on DAA prescribing in HIV/HCV co-infection.

This aim is addressed in Chapter Five.

The availability of highly effective, well tolerated interferon-free DAA regimens for HCV

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should diminish barriers to therapy in HIV/HCV co-infection. However, treatment of HIV/HCV co-infected individuals will require an awareness of the potential drug-drug interactions between specific DAAs and HIV antiretroviral agents in order to prevent morbidity and ensure treatment efficacy. Similarly in designing future pragmatic clinical trials, optimal DAA regimen choice should be considered.

In a real-world cohort of HIV/HCV co-infected individuals in Sydney, Australia, a simulation of potential drug-drug interactions between participants’ cART and interferon-free DAA regimens was performed using www.hep-druginteractions.org and relevant prescribing information. There was significant potential for drug-drug interactions between currently prescribed cART and approved interferon-free DAA regimens. In individuals on cART with HCV genotype 1 and 4 (n=128), category 3 drug-drug interactions (contra-indicated or not recommended) were noted in 0% with sofosbuvir/ledipasvir, 0% with sofosbuvir plus daclatasvir, 17% with sofosbuvir/velpatasvir, 36% with ombitasvir/paritaprevir/ritonavir +/- dasabuvir, 51% with grazoprevir/elbasvir and 51% with sofosbuvir plus simeprevir; current cART regimens were suitable for co-administration in 100%, 100%, 73%, 64%, 49% and 49%, respectively. In individuals with HCV genotype 2 or 3 (n=53), category 3 drug-drug interactions were evident in 0% with sofosbuvir plus daclatasvir, 0% with sofosbuvir and ribavirin and 13% with sofosbuvir/velpatasvir; current cART regimens were suitable in 100%, 100% and 81%, respectively.

While offering greater efficacy, tolerability and simplicity than interferon-containing regimens, drug-drug interactions will impact on DAA use and prescribing in HIV/HCV co-infection with evaluation of potential drug-drug interactions required to prevent adverse events or treatment failure. The combinations of sofosbuvir plus an NS5A inhibitor and sofosbuvir plus ribavirin appeared to be largely suitable for co-administration with commonly prescribed combination antiretroviral therapy, while drug-drug interactions frequently complicated use of HCV NS3/4a protease inhibitor-containing regimens. However, suboptimal efficacy has rendered sofosbuvir plus ribavirin largely obsolete.

Optimising direct-acting antiviral regimen choice will be crucial in future trial design to appropriately evaluate the efficacy of short duration therapy in recent HCV infection. Given favourable characteristics, including high potency and minimal drug-drug interactions, the evaluation of short duration strategy involving sofosbuvir plus an NS5A inhibitor appears to be the most appropriate.

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Implications for the findings The treatment paradigm for individuals with chronic HCV infection has evolved rapidly (228- 230, 239). The first generation HCV protease inhibitors, telaprevir and boceprevir, were approved for use in combination with pegylated-interferon and ribavirin in 2011 (358). In 2014, the first interferon-free regimens, sofosbuvir/ledipasvir and sofosbuvir plus simeprevir, were approved for the treatment of genotype 1 HCV infection. In clinical trials, combinations of two or more different classes of DAAs for eight to 12 weeks have achieved SVR in greater than 95% of treatment-naïve, non-cirrhotic individuals with chronic HCV infection (228, 230, 239).

In this context, the optimal management of recent HCV infection remains uncertain, regarding timing of treatment initiation, regimen and treatment duration (298). While in ATAHC II and DARE-C I, the majority of participants with recent HCV infection were able to receive short duration (8-16 weeks) therapy, the interferon-containing regimens used have been superseded by more tolerable oral DAA regimens. The applicability of an interferon-containing strategy in the management of HCV infection is limited to settings in which DAA therapy is unavailable or very heavily restricted.

The role and efficacy of ultra-short duration DAA therapy in recent HCV infection remains unclear. DARE-C II was one of the first studies of interferon-free DAA therapy in this population, although the regimen used is now considered suboptimal in individuals with chronic HCV genotypes 1 and 3 infection (333). Preliminary results from recent pilot studies in acute HCV infection have reported encouraging results with short duration dual-class DAA therapy, particularly in HCV mono-infection. In symptomatic acute HCV genotype 1 infection, very high SVR was demonstrated with 6 weeks of sofosbuvir/ledipasvir (SVR12 ITT 100%, 20/20);

PWID were excluded (334). Notably, baseline HCV RNA was low in the majority (≤6 log10

IU/mL, 90%; ≤3 log10 IU/mL, 45%). Again, in acute HCV genotype 1 mono-infection (PWID included), high SVR was demonstrated with 4 weeks of sofosbuvir/ledipasvir (SVR12 ITT 100%, 14/14) and 8 weeks of sofosbuvir plus simeprevir (SVR12 ITT 87%, 13/15) (335). In keeping with DARE-C II, viral suppression was rapid with HCV RNA below the limit of detection in 93% at week 1 in both arms. Lower SVR was demonstrated among individuals with HIV/HCV co-infection; SVR12 ITT was 77% (20/26) following 6 weeks of sofosbuvir/ledipasvir for acute HCV genotypes 1 and 4 (SVR12 per-protocol analysis: 83% [20/24]) (336). Consistent with DARE-C II, the three participants with relapse had high baseline HCV RNA (>6.9 log10 IU/mL), suggesting that even with potent DAA regimens, viral suppression may be protracted in those with a high HCV burden. Combined, these data support further research with contemporary dual or triple class DAA regimens in acute, particularly asymptomatic, HCV infection.

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In order to robustly evaluate the role, efficacy and cost-effectiveness of ultra-short duration DAA therapy in recent HCV infection, optimal regimen choice will be critical. Mathematical modelling suggests that rapid on-treatment second-phase viral decline could permit shorter treatment durations; very fast second-phase declines are seen following administration of HCV NS3/4A protease inhibitors and NS5A inhibitors, but not nucleoside analogues (188, 359, 360). However, drug-drug interactions will impact on DAA use in HIV/HCV co-infection. The combination of sofosbuvir plus an NS5A inhibitor appears to be suitable for co-administration with commonly used antiretroviral agents, making these DAA regimens appealing for use in HIV/HCV co-infection. However, the use of an HCV NS3/4a protease inhibitor-containing DAA regimen poses more challenges. In comparison with ATAHC I (2004-2009; HIV/HCV 31%) (133), a greater proportion of participants diagnosed with recent HCV infection in ATAHC II (2011-2015; HIV/HCV 62%), DARE-C I (2013-2015; HIV/HCV 79%) and DARE- C II (2014-2016; HIV/HCV 74%) were HIV-positive, while the proportion reporting injection drug use remained the same, highlighting the changing patterns of HCV transmission and populations at-risk of HCV acquisition in Australia.

One challenge to HCV control and elimination through therapeutic intervention is reinfection. The incidence of reinfection following treatment for recent HCV infection reported in this analysis is consistent with previous studies amongst HIV-positive MSM and PWID (reinfection incidence: 9.6 – 15.2 per 100 py) (269, 270, 285) and expands upon the previous analysis limited to the predominantly HCV mono-infected ATAHC I cohort (285). The higher incidence of HCV reinfection in this and other acute HCV cohorts contrasts with the majority of published studies in individuals treated for chronic HCV infection (reinfection incidence: 1 - 5 per 100 py).

The risk of HCV reinfection following treatment appears to be higher in those who report ongoing behaviour facilitating HCV transmission. Among PWID treated for chronic HCV infection who reported injection drug use post treatment, reinfection incidence ranged between 0.0 – 33.0 per 100 py (246, 274, 275, 281, 285-288). Similarly, in this cohort treated for recent HCV infection, reinfection incidence was significantly higher amongst participants who reported injection drug use during follow up as compared with those who did not. However, reinfection was not associated with injection drug use prior to or at commencement of therapy. Particularly in the setting of interferon-based therapy, there may have been considerable selection bias in those PWID deemed suitable, or willing, for treatment. While injecting risk behaviour among PWID appeared to decline during and after interferon-based treatment (338), it is possible that expanded HCV treatment access and DAA therapeutic optimism may be

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associated with increased risk behaviour, as seen among MSM following the introduction of HIV combination antiretroviral therapy (339).

The increased risk of reinfection seen with injection drug use post treatment in association with use of unsterile needles and syringes highlights the need for education and broad access to harm reduction and prevention strategies in concert with HCV treatment. For PWID, access to interventions known to prevent HCV infection, including OST and high coverage needle and syringe access programs (40, 47-49, 340), will be crucial to minimise reinfection risk. Education regarding the potential for reinfection by health providers, peer-support workers and community drug user organisations should be offered. Screening protocols for acute HCV in PWID and HIV-positive MSM should be employed to enhance early HCV diagnosis (91, 103, 295).

As exemplified in this cohort, populations at high risk of reinfection, such as PWID and HIV- positive MSM, are not mutually exclusive. HIV-positive MSM who inject drugs are at significantly higher risk of HCV reinfection than HIV-positive MSM who do not inject drugs. While often discussed as separate cohorts, it is important to remember that there is significant overlap. However, different drug use behaviours reported by cohorts of HIV-positive MSM as compared with HCV mono-infected populations may necessitate different public health strategies, with the predominance of amphetamine use among HIV-positive MSM who inject drugs a particular feature. Ongoing risk behaviours associated with HCV transmission and high HCV reinfection rates may compromise the population-level benefits of Treatment-as- Prevention among recent PWID and HIV-positive MSM commencing DAA therapy.

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Directions for future research The development of highly effective, well tolerated interferon-free DAA therapy ushers in a time of great optimism and provides the therapeutic tools required to strive for HCV eradication. In order to achieve this goal, strategies to curb ongoing transmission are required.

One approach to reducing transmission is enhancing diagnosis and treatment of recent HCV infection. As such, the role of interferon-free DAA therapy in the management of recent HCV infection requires further evaluation. It is uncertain whether acute or recent HCV infection will continue to offer a unique therapeutic advantage, with regard to shortened treatment duration as compared with chronic HCV infection. For most individuals with chronic HCV infection, eight to 12 weeks of dual-class DAA therapy is suitable. With continued advances in HCV therapeutics, six weeks of second-generation dual- or triple-class DAA therapy may be appropriate for non-cirrhotic individuals, although four weeks still appears to be too short (230).

Optimising DAA regimen choice will be crucial in future trial design to appropriately evaluate the efficacy of short duration therapy in recent HCV infection. Given mathematical modelling which suggests that rapid on-treatment second-phase viral decline could permit shorter treatment durations, the use of a potent dual- or triple-class DAA regimen which includes an HCV NS3/4A protease inhibitor and/or NS5A inhibitor (with or without a nucleotide analogue) appears justified (188, 359, 360).

Clinical trials examining the efficacy of short duration interferon-free dual or triple class DAA therapy have commenced; six weeks of sofosbuvir/ledipasvir in symptomatic acute HCV genotype 1 mono-infection yielded excellent results (334). A summary of registered interferon- free clinical trials in recent HCV infection is presented in Table 6-1. DARE-C II has confirmed the feasibility of ultra-short DAA therapy and informed future trial design in this population. TARGET3D (NCT02634008) is examining the efficacy and safety of paritaprevir/ritonavir/ombitasvir and dasabuvir (with or without ribavirin) for six or eight weeks in recent genotype 1 HCV infection, both primary and reinfection; this trial is open to recruitment in Australia, New Zealand and England. REACT (NCT02625909), an international randomised control trial, will examine the efficacy and safety of ultra-short (six weeks) versus standard duration (12 weeks) sofosbuvir/velpatasvir for recent HCV infection (n=250). The robust study design, large sample size, use of potent pan-genotypic DAA therapy and inclusion of both primary infection and reinfection should greatly enhance our understanding of the utility of DAA therapy in recent HCV infection.

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Table 6-1. Registered interferon-free DAA clinical trials in recent HCV infection

Country, Duration of Treatment PI +/- study Status year Short title infection Study population N Regimen duration group (SVR12*) commenced (months) (weeks) Australia, Matthews G; DARE-C II GT 1-6; HIV-positive Closed to recruitment New Zealand ≤12 19 SOF+RBV 6 Kirby Institute NCT02156570 and PWID eligible (6/19; 32%) 2014 Chung RT, HCV GT 1-6 and US SWIFT-C 17 SOF+RBV Arm A: 12 Recruiting (Arm B) Naggie S; ≤6 HIV-positive; PWID 2014 NCT02128217 27 LDV/SOF Arm B: 8 (Arm A: 10/17; 59%) ACTG eligible HepNet Acute HCV GT 1; HIV- Wedemeyer H; Germany Closed to recruitment HCV IV ≤4 positive and PWID 20 LDV/SOF 6 HepNet 2014 (20/20; 100%) NCT02309918 ineligible Germany, GT 1 or 4 and HIV- Closed to recruitment Rockstroh J UK NCT02457611 ≤6 positive; PWID 26 LDV/SOF 6 (20/26; 77%) 2015 eligible Australia, Matthews G; New Zealand, TARGET3D GT 1; HIV-positive PrOD +/- Arm A: 8 ≤12 60 Recruiting Kirby Institute UK NCT02634008 and PWID eligible RBV Arm B: 6 2016 Netherlands, GT 1 or 4 and HIV- DAHHS-2 Rijnders B Belgium ≤6 positive; PWID 80 GZR/EBR 8 Recruiting NCT02600325 2016 eligible

Matthews G; International REACT GT 1-6; HIV-positive 6 or 12 ≤12 250 SOF/VEL Not yet recruiting Kirby Institute 2016 NCT02625909 and PWID eligible (RCT 1:1)

*SVR12 if applicable Abbreviations: EBR, elbasvir; GBR, grazoprevir; GT, genotype; LDV, ledipasvir; N, number of participants enrolled or to be enrolled; PI, Principal Investigator; PrOD, paritaprevir/ritonavir/ombitasvir + dasabuvir; RBV, ribavirin; RCT, randomised control trial; SOF, sofosbuvir

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While pilot studies with ultra-short duration sofosbuvir/ledipasvir in acute genotype 1 HCV demonstrated promising efficacy (334-336), many questions remain to be answered. The impact of clinical, virological and immunological factors on treatment outcome, such as HIV infection, baseline HCV RNA, mode of HCV transmission (injection drug use, sexual exposure), clinical presentation and duration of infection, remain to be adequately determined. In the era of DAA therapy, it is uncertain whether acute HCV infection will offer a therapeutic advantage in comparison with chronic HCV infection. The impact of duration of HCV infection on DAA efficacy is unclear and requires further investigation. In the study by Deterding et al (334), duration of infection at screening was short (≤4 months), while in the study by Rockstroh et al (336), the protocol specified a duration of infection ≤6 months. However, the estimated duration of HCV infection could not be determined for all participants, raising the possibility that treatment commencement occurred in the early chronic phase of infection. DARE-C II permitted enrollment of individuals with an estimated duration of HCV infection of ≤12 months. As the distinction between acute and early chronic infection is somewhat arbitrary, further research regarding timing of treatment initiation within one year of infection is warranted and would allow for broader clinical application. These pilot studies offer exciting potential, but are limited by small sample sizes and selected populations.

Baseline HCV RNA and early on-treatment viral kinetics may influence response to short duration DAA therapy in recent HCV infection, with higher baseline HCV RNA and more protracted HCV RNA suppression associated with post-treatment relapse (335-337). Given the potential for high fluctuating HCV RNA in recent infection (102), a shortened duration therapeutic strategy may require stratification by baseline HCV RNA. There is precedent for this approach in chronic HCV infection. While acknowledging the statistical limitations, post hoc analysis of baseline HCV RNA in the ION-3 trial formed the basis for prescribing guidelines regarding shortening the treatment duration of sofosbuvir/ledipasvir to eight weeks in treatment-naïve non-cirrhotic individuals with chronic HCV genotype 1 and baseline HCV RNA <6 000 000 IU/mL (324, 361, 362). A recent pilot study in China evaluated ultra-short duration response-guided triple-class DAA therapy among treatment-naïve non-cirrhotic individuals with chronic genotype 1b HCV infection (363). Very high SVR (100%; 18/18) was seen among those who received three weeks of DAA therapy after achieving an ultra-rapid viral response (defined as HCV RNA <500 IU/mL after 48 hours). Participants with an ultra-rapid viral response had a significantly lower mean baseline HCV RNA than those without an ultra- rapid viral response (HCV RNA 6.0 log10 IU/mL [SD 0.8] vs 7.0 log10 IU/mL [SD 0.3]; p<0.001), suggesting that baseline HCV RNA could assist in predicting which individuals may respond favourably to short duration therapy.

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While individualised therapy contributes to complexity, it may be possible to develop a simple clinical algorithm to determine the likelihood of achieving SVR with ultra-short duration therapy in a given individual with recent HCV infection. The impact of key clinical, immunological and virological parameters, such as HIV infection, baseline HCV RNA, mode of HCV transmission, clinical presentation and duration of infection, on response to DAA therapy in recent HCV infection should be further assessed. While administration of interferon in acute HCV infection appeared to play a unique role in enhancing outcome due to differences in expression of interferon-stimulated genes (139), the impact of host genetics and immune response on treatment outcome with DAA therapy in recent HCV infection is unknown. While shorter treatment durations should reduce cost and enhance adherence, many questions remain to be answered regarding the role of DAA therapy in recent HCV infection.

In addition to treating acute HCV infection, DAA treatment scale-up and elimination of HCV in specific high-risk populations should assist in eradication. Mathematical modelling suggests that substantial reductions in HCV incidence and prevalence could be achieved by targeted DAA treatment scale-up amongst those at highest risk of ongoing transmission, including PWID and HIV-positive MSM (75, 271, 291, 292). To date, most HCV clinical trials have largely excluded those at highest risk of transmission, including individuals with recent HCV infection and individuals reporting recent injection drug use. Further evidence is required in these populations to assess DAA efficacy, long term outcomes, and crucially, reinfection incidence.

The incidence of HCV reinfection following DAA-based treatment is unknown and needs careful evaluation as access to treatment among populations at risk of ongoing transmission increases. There is some uncertainty around current reinfection estimates following interferon- based therapy given small sample sizes, retrospective study designs, exclusion of recent PWID from clinical trials, varied definitions for recent injection drug use and time at-risk for reinfection, and the inability to accurately distinguish relapse from reinfection. Sufficient follow-up time post treatment, with HCV RNA testing at regular intervals (3-6 monthly), will be required to appropriately evaluate reinfection incidence. While injecting risk behaviour among PWID appeared to decline during and after interferon-based treatment (338), it is possible that expanded HCV treatment access and DAA therapeutic optimism may be associated with increased risk behaviour, as seen among MSM following the introduction of HIV combination antiretroviral therapy (339). Factors associated with reinfection following treatment will need to be assessed, with robust methodology, and retreatment strategies evaluated. In treating populations at high risk of transmission and reinfection, a prophylactic vaccine or long-acting depot preparations (for example, microRNA-122 inhibitors; see

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Supplementary Appendix Table1-1) could be of particular benefit. In addition, enhanced surveillance will be required to assess the population-level impact of DAA treatment scale-up.

With DAA treatment scale-up among populations at high risk of transmission, implementation and evaluation of novel prevention strategies should be a priority. Populations at risk of HCV acquisition, such as PWID and HIV-positive MSM, are not mutually exclusive. However, different drug use and sexual behaviours within these populations have implications for public health strategies. Much of the evidence for HCV prevention among PWID focusses on individuals who inject opiates (40, 47-49, 340). Increasing use of stimulant drugs (largely amphetamines) and the phenomena of ‘chemsex’ (illicit drug use before or during sex, by both injecting and non-injecting routes of administration) may necessitate a different approach in MSM (77, 80-83). Evidence supporting sexual behavioural interventions for HCV prevention among MSM is lacking. A comprehensive approach to HCV care involving the combination of DAA treatment and behavioural intervention is being trialled Among HIV-positive MSM in the Swiss Cohort Study (“The Swiss HCVfree trial”, NCT02785666). The behavioural intervention involves targeted counselling about sexual risk behaviour and recreational drug use in participants reporting inconsistent condom use with casual male partners. All participants receive “standard of care” (written and oral) information regarding the risk of HCV reinfection following successful treatment. With increasing use of HIV pre-exposure prophylaxis, there is the potential for a reduction in sero-sorting of sexual partners, increased sexual risk behaviour and transmission of HCV among HIV-positive and HIV-negative MSM populations (77, 86, 87). Additionally, use of social media sexual networking applications has been associated with the increase in HCV incidence among HIV-positive MSM; this could be utilised to direct targeted messaging to this population regarding HCV testing, diagnosis, treatment and prevention.

Greater HCV testing, diagnosis and linkage to care is required to facilitate DAA treatment scale-up. Novel methods of service delivery to marginalised populations could enhance DAA treatment uptake. Screening protocols for acute HCV in specific high-risk populations, including young PWID (35, 104, 105), incarcerated PWID (33, 106) and HIV-positive MSM (58, 107), should be considered (310, 364), utilising rapid or point-of-care diagnostics (365- 368). The advent of daily pan-genotypic fixed-dose combination DAA therapy signals a major advance in HCV therapeutics (228, 333). Rapid diagnostics coupled with the ability to deliver safe short duration pan-genotypic DAA therapy could maximise acceptability to both the patient and prescriber, by providing effective, simple HCV care and treatment, which should require little efficacy and safety monitoring and would allow for HCV management outside of traditional tertiary centres. The feasibility of a “test and treat” strategy utilising point-of-care

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diagnostics with simplified monitoring could be examined in an effort to enhance treatment uptake in marginalised populations with a high burden of HCV disease.

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Thesis strengths and limitations The work presented in this thesis stems from analysis of five multi-centre prospective trials in Australia and New Zealand, involving marginalised populations, including people with recent HCV infection, people who inject drugs and people with HIV/HCV co-infection, and spans a period of marked transition in the field of HCV therapeutics. The research themes discussed are of importance in the emerging era of interferon-free DAA therapy, as HCV treatment access expands to populations at high risk of transmission and reinfection.

Sample size and the applicability of the regimens used may be considered study limitations in ATAHC II, DARE-C I and DARE-C II. Given inherent difficulties in identifying those with recent HCV infection, sufficient sample size can be difficult to obtain. The respective sample sizes of the reported clinical trials limited the power of the statistical analysis, impacting upon the strength of conclusions drawn. Although ATAHC II is one of the larger studies in recent HCV infection, the sample size meant that Cox proportional hazards and logistic regression analyses were limited to assessment of key virological and treatment factors. Both DARE-C I and DARE-C II were pilot studies, designed to evaluate feasibility, adverse events and effect size, in an attempt to predict appropriate sample size and improve upon the study design of larger forthcoming clinical trials. Despite the small enrolled populations, both DARE-C I and DARE-C II provided conclusive results, namely the considerable toxicity associated with PEG- IFN, ribavirin and telaprevir in DARE-C I and the suboptimal efficacy of sofosbuvir and ribavirin for only six weeks in DARE-C II.

Given the pace of change in HCV therapeutics, the interferon-containing regimens used in ATAHC II and DARE-C I and the interferon-free DAA regimen in DARE-C II have been rapidly superseded by more potent combination DAA regimens. The speed of DAA development coupled with the known adverse event profile of interferon contributed to the early closure of ATAHC II; study termination did not impact upon the primary outcome of the trial. Despite these limitations, combined, these studies confirmed the feasibility of short duration therapy in those with recent HCV infection, regardless of treatment regimen, and have informed future trial design and implementation.

Limitations inherent to the statistical methodology require discussion, particularly in light of the sample size and number of events. The ‘rule of thumb’ that logistic and Cox models should be used with a minimum of 10 events per predictor variable may be overly conservative; depending on the scenario, five to nine events per predictor variable may be reasonable (316). However, potential type I and II error need to be considered. Acknowledging the possibility of type II error, misleading conclusions can usually be avoided if negative findings are interpreted

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in the light of confidence intervals. Overall, when a statistically significant association is found in a model with less than 10 events per predictor variable, a degree of caution is warranted, but plausible and highly significant associations hypothesized a priori should not be ignored (316), as was the case in the logisitic and Cox analyses performed for ATAHC II, with only one significant variable reported for each model. Additionally, with small sample sizes, it can be difficult to appropriately select variables for inclusion in a logistic regression model. Stepwise selection with a low α can lead to relatively poor model performance (369). Backwards stepwise elimination may not preserve nominal statistical properties and may be misleading. There is support for other selection methods including standard maximum likelihood, use of a linear shrinkage factor, penalized maximum likelihood, or quantitative external information on univariable regression coefficients (369). In small data sets, better performance may be obtained using full models with a limited number of important predictors, where regression coefficients are reduced with any of the shrinkage methods (369). However, use of stepwise elimination did not change the statistical output in this case.

The analysis of HCV reinfection following treatment for recent HCV infection has a number of strengths, including the prospective design, inclusion of active PWID and HIV/HCV co-infected MSM, robust definition of follow up time at-risk for reinfection and use of viral sequencing to accurately delineate relapse and reinfection. The inclusion of a relatively large at-risk population, and documentation of ten cases of reinfection, provided an opportunity to explore associations, but the sample size and number of reinfection cases did limit the strength of the conclusions drawn. The statistical methodology used to assess for factors associated with reinfection (exact Poisson regression) attempted to address some of the issues related to sample size and event number. The Poisson regression model fits the assumptions of the underlying random process generating a small number of events at a rate (eg, number [reinfection] per unit time [years at risk]) determined by other variables in the model. Using exact Poisson regression, the confidence intervals are expected to be wide; point estimates are not expected to be precise. This highlights the uncertainty inherent to the methodology, particularly in light of the sample size and number of events. The analysis was intended to be hypothesis-generating, raising awareness of factors which may impact upon the population-level benefits of DAA treatment scale-up. Inherent limitations in the original clinical trials impacting on the analysis included the duration of follow up post treatment, the lack of sexual behaviour data collection and the small numbers receiving interferon-free DAA therapy. Sufficient follow-up time post treatment, with HCV RNA testing at regular intervals, will be required to appropriately evaluate reinfection incidence. The incidence and risk factors associated with HCV reinfection following DAA-based treatment will need careful evaluation with robust methodology as access to treatment among populations at-risk of ongoing transmission increases.

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Conclusion The advent of highly effective, well tolerated interferon-free DAA therapy has revolutionised HCV therapeutics (140). Combining two or more potent DAAs from different classes has increased SVR (>90%) and shortened treatment duration to only eight to 12 weeks in most populations with chronic HCV infection (228, 230, 239). While excellent results in chronic HCV infection have cast doubt on any “efficacy advantage” of early treatment in acute HCV infection (140), diagnosis and treatment of recent HCV infection should facilitate engagement in multidisciplinary care, prevent the development and complications of chronic liver disease and curb ongoing transmission in key populations (75, 300). The role of ultra-short duration DAA therapy in recent HCV infection requires further evaluation.

The burden of disease attributed to HCV is high among PWID, and is increasing among HIV- infected MSM. The potential for broad access, rapid DAA treatment scale-up and further treatment simplification have stimulated discussion around HCV Treatment-as-Prevention and HCV elimination. HCV Treatment-as-Prevention strategies will be enhanced by early diagnosis and increased treatment uptake in recent HCV infection (75, 291). Ultimately, the population- level impact of DAA therapy will relate to facilitating global access to HCV screening, care and treatment. Overcoming barriers imposed by high drug pricing, drug use and liver disease stage restrictions and stigma will be central to this (307). The significant risk for HCV reinfection following treatment in individuals with ongoing behaviour facilitating HCV transmission emphasises the need for post-treatment surveillance, harm reduction strategies and education (270, 311), but must not be considered an impediment to treatment, if HCV elimination is to be achieved.

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Supplementary Appendix

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Investigational direct-acting antivirals for the treatment of HCV infection.

Supplementary Appendix Table 1.1. Investigational HCV DAAs in Phase II and III development HCV Drug Company Comments GT NS3/4A Protease Inhibitor Glecaprevir Phase III, oral formulation 1-6 AbbVie (ABT-493) Co-formulated with pibrentasvir Voxilaprevir Phase III, oral formulation 1-6 Gilead (GS-9857) Co-formulated with sofosbuvir and velpatasvir Vedroprevir 1 Gilead Phase II, oral formulation (GS-9451) NS5B RdRp Inhibitor – nucleotide analogue Alios BioPharma AL-335 1-6 Phase II, oral formulation (Janssen) Phase II, oral formulation MK-3682 1-6 Merck & Co Co-formulated with grazoprevir and MK-8408 NS5A Assembly Inhibitor Phase II, oral formulation MK-8408 1-6 Merck & Co Co-formulated with grazoprevir and MK-3682 Odalasvir 1-6 Achillion Phase II/III, oral formulation (ACH-3102) Pibrentasvir Phase III, oral formulation 1-6 AbbVie (ABT-530) Co-formulated with glecaprevir Phase III, oral formulation Ravidasvir Pharco 1-6 Being developed for use in combination with (PPI-668) Pharmaceuticals sofosbuvir MicroRNA-122 Inhibitor Phase IIa, subcutaneous formulation Miravirsen 1-6 Santaris Pharma. Modified oligonucleotide antagonist of miR-122 Phase IIa, subcutaneous formulation Regulus RG-101 1-6 Modified oligonucleotide antagonist of miR-122 Therapeutics conjugated to N-acetylgalactosamine Cyclophilin Inhibitor Phase IIb/III, oral formulation Alisporivir Non-immunosuppressive cyclosporin A 1-6 Novartis (Debio-025) analogue - inhibition of cyclophilin A with modulation of NS5A function Abbreviations: RdRp, RNA-dependent RNA polymerase

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Further information regarding specific investigational DAAs.

NS3/4A Protease Inhibitors Glecaprevir (ABT-493) is a potent second-generation pan-genotypic HCV protease inhibitor, co-formulated with pibrentasvir (ABT-530) (156). In Phase II studies, very high SVR12 (97- 100%) was achieved with eight to 12 weeks of glecaprevir/pibrentasvir 300/120mg daily in HCV genotypes 1-6, including participants with genotype 3 and compensated cirrhosis (147, 370). High SVR12 (91%) was also achieved in DAA-experienced HCV genotype 1 non- cirrhotic participants with 12 weeks of glecaprevir/pibrentasvir 300/120mg daily plus ribavirin (371). Glecaprevir/pibrentasvir appears to be safe and well tolerated; the most commonly reported adverse events were fatigue (18%), headache (17%), nausea (13%) and diarrhoea (10%) (372). The combined oral formulation and dose will be: glecaprevir/pibrentasvir 100/40mg, three tablets daily. FDA-approved in the third quarter of 2017 is expected.

Voxilaprevir (GS-9857). This drug is a pan-genotypic second-generation NS3/4A protease inhibitor undergoing Phase III clinical trials as part of an oral once daily fixed dose combination DAA regimen co-formulated with sofosbuvir (a nucleotide polymerase inhibitor) and velpatasvir (NS5A assembly inhibitor) as sofosbuvir/velpatasvir/voxilaprevir 400/100/100 mg. Phase II efficacy and safety results were encouraging with short duration therapy (NCT02202980) (230). Among treatment naïve cirrhotic and non-cirrhotic participants with HCV genotypes 1 and 3, SVR12 ranged between 83-93% following only six weeks of sofosbuvir/velpatasvir/voxilaprevir. In treatment-experienced (DAA naive) cirrhotic and non- cirrhotic participants with HCV genotypes 1 and 3 receiving eight weeks of sofosbuvir/velpatasvir/voxilaprevir, SVR12 was 100%, falling to 89% in participants with HCV genotype 1 who had failed protease inhibitor-based triple therapy. Small numbers of participants who had failed an oral interferon-free regimen were included; six weeks of sofosbuvir/velpatasvir/voxilaprevir was suboptimal in genotype 1 (SVR12 67%; 20/30), but eight weeks was more promising in genotype 3 (SVR12 100%; 4/4). The most commonly reported adverse events were headache, nausea, and fatigue (230). Phase III trial results comparing sofosbuvir/velpatasvir/voxilaprevir (eight or 12 weeks) with sofosbuvir/velpatasvir (12 weeks) are expected later in 2016, including DAA-treatment experienced participants.

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Vedroprevir (GS-9451). This HCV NS3/4a protease inhibitor is being developed as an oral formulation in current Phase II trials, for administration in combination with an NS5B RdRp inhibitor and NS5A inhibitor. Combination DAA regimens using potent agents from two or three different classes may further reduce treatment duration. In participants with HCV genotype 1 infection without cirrhosis, SVR12 was achieved in 95% (19/20) following six weeks of sofosbuvir/ledipasvir plus vedroprevir (373). In participants with HCV genotype 1 infection and cirrhosis, SVR12 was achieved in 88-95% following eight weeks of sofosbuvir/ledipasvir plus vedroprevir with (21/24) or without (21/22) ribavirin (374). Further clinical development of vedroprevir is uncertain given the large Phase III clinical program combining voxilaprevir with sofosbuvir and velpatasvir.

NS5B RdRp Inhibitor – nucleotide analogues AL-335. One of a small number of nucleotide analogue NS5B RNA-dependent RNA polymerase inhibitors being studied or used (e.g. sofosbuvir), AL-335 is a potent pan-genotypic uridine analogue with EC50 values ranging from 0.04 to 0.07 uM (HCV genotypes 1-6). AL- 335 was well tolerated following single and multiple doses with pharmacokinetics supporting daily dosing. Following seven days of 800mg daily, the maximal mean reduction in HCV RNA in participants with and without cirrhosis was 2.75 – 4.75 log10 IU/mL. Several Phase 2 trials are due to start recruiting in 2016 assessing the combination of short duration (six or eight weeks) combination DAA therapy with AL-335, odalasvir +/- simeprevir (clinicaltrials.gov NCT02569710, NCT02765490). AL-335 could be an important competitor for Gilead’s sofosbuvir nucleotide. (151, 152)

MK-3682 is a pan-genotypic nucleotide analogue NS5B RdRp inhibitor which is in Phase II clinical development. Seven-day monotherapy trials with MK-3682 showed potent activity for both HCV genotype 1a and 1b infected subjects, with mean maximum viral load reductions in the 300 mg arms of 4.8 log10 IU/ml for genotype 1a and 3.9 log10 IU/ml for genotype 1b.

Similar results for 300 mg arms were seen for genotype 2- (4.6 log10 IU/ml) and genotype 3- infected subjects (4.1 log10 IU/ml). In Phase II trials, high SVR12 has been seen with eight weeks of MK-3682 300mg or 450mg + grazoprevir 100mg + MK-8408 60mg was evaluated in HCV genotypes 1-3 treatment-naïve non-cirrhotic participants (153). In participants receiving MK-3682 300mg daily, SVR12 was 100% (24/24) in genotype 1, 71% (10/14) in genotype 2 and 95% (20/21) in genotype 3, while in participants receiving MK-3682 450mg daily, SVR12 was 91% (21/23) in genotype 1, 94% (15/16) in genotype 2 and 91% (20/22) in genotype 3. The pan-genotypic triple DAA combination appeared to be safe and well tolerated. Further clinical development is being pursued with MK-3682 450mg daily. Phase II trials are ongoing with MK3682B, the fixed dose combination of MK-3682/grazoprevir/MK-8408 (dose:

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MK3682B 225/50/30mg, two tablets daily), in other populations, including HCV genotypes 4-6, HIV/HCV co-infection and DAA-treatment experienced.

NS5A Assembly Inhibitors MK-8408 is a second-generation NS5A assembly inhibitor in Phase II clinical trials for oral administration co-formulated with grazoprevir (NS3/4A protease inhibitor) and MK-3682 (nucleotide analogue NS5B RdRp inhibitor) (375, 376). See Paragraph MK-3682 for further details.

Odalasvir (ACH-3102). This second-generation NS5A assembly inhibitor is in Phase II clinical trials as an oral formulation. It retains activity against HCV variants resistant to first generation NS5A inhibitors, including ledipasvir and daclatasvir (377). A Phase IIa open-label randomized partial-crossover study evaluated the efficacy, safety, and tolerability of 6 or 8 weeks of odalasvir 50mg daily with sofosbuvir 400mg daily for treatment-naïve genotype 1 HCV- infected patients (377, 378). 30 patients were enrolled, with 24 randomised to treatment. Among those randomized to 6 or 8 weeks treatment, 71% had HCV genotype 1a and mean baseline HCV RNA was 7.0 log10 IU/mL (range 5.5 – 8.0 log10 IU/mL). In cohort 1, 12 patients completed 8 weeks of treatment; SVR12 was 100%. Subsequently, in cohort 2, 12 patients were treated for 6 weeks; SVR12 was also 100%. Odalasvir and sofosbuvir were well tolerated with no significant adverse events, ECG findings, or lab abnormalities observed during treatment. Several Phase IIb trials are due to commence recruitment in 2016 assessing the safety and efficacy of short duration (six or eight weeks) therapy with AL-335, odalasvir +/- simeprevir (clinicaltrials.gov NCT02569710, NCT02765490).

Pibrentasvir (ABT-530). An orally-administered pan-genotypic NS5A assembly inhibitor. See Paragraph ABT-493 for further details (147, 156, 370-372)

Ravidasvir (PPI-668). This pan-genotypic NS5A assembly inhibitor is being developed for use in combination with sofosbuvir. Conducted in Egypt, a large phase III registration trial in individuals with HCV genotype 4 infection demonstrated SVR12 ranging from 86-100% with 12 or 16 weeks of sofosbuvir (400mg daily) and ravidasvir (200mg daily), with or without ribavirin (155). Of the 300 genotype 4 infected subjects, nearly 70% were men and the mean age was approximately 48 years. Half were treatment naive & half had received prior interferon- based therapy. More than 40% had compensated liver cirrhosis. Participants were stratified according to prior treatment and cirrhosis status. Participants in 3 groups received 200mg ravidasvir & 400mg sofosbuvir once daily for 12 weeks, and were randomly assigned to either add ribavirin or not. The harder-to-treat patients in group 3 (interferon treatment-experienced

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with cirrhosis) received ravidasvir plus sofosbuvir and ribavirin for either 12 or 16 weeks. Among participants without cirrhosis treated with ravidasvir and sofosbuvir alone, 100% of previously untreated and 95% of treatment-experienced subjects achieved SVR12, while among those who added ribavirin, SVR12 rates were 98 and 100%, respectively. In treatment-naive participants with cirrhosis, SVR12 rates were 93% with ravidasvir and sofosbuvir alone and unchanged (92%) with the addition of ribavirin. Among the treatment-experienced participants with cirrhosis, only 86% were cured with ravidasvir, sofosbuvir and ribavirin taken for 12 weeks, but the SVR12 rate rose to 100% when treatment was extended to 16 weeks (379).

MicroRNA-122 Inhibitors Miravirsen (SPC3649). This drug is a modified oligonucleotide which antagonises the liver- expressed micro-RNA (miR-122). Propagation of HCV is dependent on a functional interaction between the HCV genome and the liver-expressed miR-122; miravirsen sequesters mature miR- 122 in a highly stable heteroduplex, thereby disrupting that dependency. Because miravirsen is an oligonucleotide, not absorbed orally, it is formulated for subcutaneous injection. A Phase IIa randomized, double blind, placebo-controlled, ascending multiple dose-ranging study enrolled 36 subjects with chronic HCV genotype 1 between September 2010 and November 2011 who were given 5 weekly injections of miravirsen for 4 weeks at doses of 3, 5 or 7 μg; treatment with miraversin resulted in prolonged dose dependent reduction in HCV RNA by 1.2 to 3.2 log10 as compared with placebo (-0.4 log10). . As compared with placebo, participants receiving miravirsen reported no significant dose-related adverse reactions and there was no evidence of resistant virus appearing (158, 160, 380-382).

RG-101 is similar to miravirsen in that it is a modified oligonucleotide that is an antagonist of miR-122, but it is conjugated to N-acetylgalactosamine which facilitates RG-101 hepatocyte uptake and increases its potency approximately 20-fold. Like miravirsen, monotherapy with RG-101 dramatically reduced HCV RNA when given subcutaneously on a weekly basis. A Phase II study of RG-101 in combination with sofosbuvir/ledipasvir, simeprevir or daclatasvir is ongoing (closed to recruitment). 79 treatment-naïve non-cirrhotic HCV subjects with either genotype 1 or 4 received a four-week treatment regimen containing a subcutaneous administration of 2 mg/kg of RG-101 at day 1 and day 29, in combination with 4 weeks of once daily approved anti-viral agents sofosbuvir/ledipasvir (n=27), simeprevir (n=27) and or daclatasvir (n=25). Interim results were presented in April 2016 with SVR12 100% (14/14), 93% (14/15) and 100% (12/12) in subjects receiving sofosbuvir/ledipasvir, simeprevir and daclatasvir, respectively; final results are expected in late 2016. On 27 June 2016 RG-101’s sponsor announced, “[we] received oral notice from the U.S. Food and Drug Administration (FDA) that [its] IND for RG-101 had been placed on clinical hold. Regulus anticipates it will

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receive a formal clinical hold letter from the FDA within 30 days and plans to work diligently with the agency to seek the release of the clinical hold.” The clinical hold was placed following two serious adverse events with jaundice and occurred after current Phase II trials had completed enrolment (159, 160).

Cyclophilin Inhibitors Alisporivir (Debio-025) and other cyclophilin inhibitors. Cyclophilin inhibitors are orally- absorbed, non-immunosuppressive analogues of cyclosporin A, the first “host-targeting” antiviral drugs. Cyclophilin inhibitors block HCV replication by neutralizing the peptidyl- prolyl isomerase activity of the abundant, host-cytosolic protein, cyclophilin A. Since native cyclophilin A is important for HCV NS5A assembly, inhibiting it with a cyclophilin inhibitor blocks HCV replication by blocking NS5A functions. Due to their unique mechanism of antiviral action, cyclophilin inhibitors are pan-genotypic, provide a high barrier for development of viral resistance, are active against all common resistance associated substitutions, and demonstrate additive or synergistic effects in vitro with approved DAAs. Cyclophilin inhibitors generally have good pharmacokinetic and safety profiles. Phase I and II clinical studies have demonstrated that cyclophilin inhibitors dramatically reduce viral loads in HCV-infected patients.

Phase III studies have been conducted with alisporivir in treatment-naïve participants with HCV genotypes 1, 2 and 3. In participants with HCV genotype 1 (n=1081), alisporivir (600mg daily or 400mg twice daily) was administered in combination with response-guided pegylated interferon and ribavirin (161). Overall, SVR12 was 69% in all alisporivir groups compared with 53% in the pegylated-interferon and ribavirin control arm. The highest SVR12 (90%) was achieved in participants treated with alisporivir 400 mg twice daily plus pegylated-interferon and ribavirin for greater than 24 weeks. In participants with HCV genotype 2 and genotype 3 (n=340), alisporivir (600 – 1000 mg daily) was administered in combination with ribavirin and/or pegylated interferon. SVR24 (ITT) in the alisporivir arms ranged from 80% - 85%, compared with 58% in the pegylated-interferon plus ribavirin arm. Viral breakthrough occurred, though was infrequent (3%; n = 7 of 258). The most frequent clinical and laboratory adverse events associated with alisporivir in combination with pegylated interferon-α and ribavirin were similar to those associated with pegylated interferon-α and ribavirin used alone. While these interferon-containing strategies will not be utilised, alisporivir may be explored in combination with interferon-free DAA regimens.

Other cyclophilin inhibitors have been administered in proof-of-concept or small exploratory trials, including CPI-431-32, NIM811 and SCY-635. In an in vitro assay using HIV and HCV

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co-infection, cyclophilin inhibitors, including CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In 2016, a phase I randomized, double-blind study commenced to assess the safety, pharmacokinetics and efficacy of EDP-494 in healthy volunteers and in treatment-naive subjects with HCV genotypes 1 and 3 (NCT02652377). The future of these “anti-host” drugs is uncertain (161-163, 383).

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Chemical structure of individual approved HCV direct-acting antivirals Protease inhibitors

Supplementary Appendix Figure 1-1. Chemical structure of boceprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=10324367, https://pubchem.ncbi.nlm.nih.gov/compound/10324367 (accessed Nov. 25, 2015).

Supplementary Appendix Figure 1-2. Chemical structure of telaprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=3010818, https://pubchem.ncbi.nlm.nih.gov/compound/3010818 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-3. Chemical structure of simeprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=24873435, https://pubchem.ncbi.nlm.nih.gov/compound/24873435 (accessed Nov. 25, 2015).

Supplementary Appendix Figure 1-4. Chemical structure of asunaprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=16076883, https://pubchem.ncbi.nlm.nih.gov/compound/16076883 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-5. Chemical structure of paritaprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=45110509, https://pubchem.ncbi.nlm.nih.gov/compound/45110509 (accessed Nov. 25, 2015).

Supplementary Appendix Figure 1-6. Chemical structure of grazoprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=71576667, https://pubchem.ncbi.nlm.nih.gov/compound/71576667 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-7. Chemical structure of vaniprevir From: National Centre for Biotechnology Information. PubChem Compound Database; CID= 59856471, https://pubchem.ncbi.nlm.nih.gov/compound/59856471 (accessed May. 6, 2016).

NS5B polymerase inhibitors

Supplementary Appendix Figure 1-8. Chemical structure of sofosbuvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=45375808, https://pubchem.ncbi.nlm.nih.gov/compound/45375808 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-9. Chemical structure of dasabuvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=56640146, https://pubchem.ncbi.nlm.nih.gov/compound/56640146 (accessed Nov. 25, 2015).

NS5A inhibitors

Supplementary Appendix Figure 1-10. Chemical structure of ledipasvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=67505836, https://pubchem.ncbi.nlm.nih.gov/compound/67505836 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-11. Chemical structure of ombitasvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=54767916, https://pubchem.ncbi.nlm.nih.gov/compound/54767916 (accessed Nov. 25, 2015).

Supplementary Appendix Figure 1-12. Chemical structure of daclatasvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=25154714, https://pubchem.ncbi.nlm.nih.gov/compound/25154714 (accessed Nov. 25, 2015).

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Supplementary Appendix Figure 1-13. Chemical structure of elbasvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=71661251, https://pubchem.ncbi.nlm.nih.gov/compound/71661251 (accessed Nov. 25, 2015).

Supplementary Appendix Figure 1-14. Chemical structure of velpatasvir From: National Centre for Biotechnology Information. PubChem Compound Database; CID=91885554, https://pubchem.ncbi.nlm.nih.gov/compound/91885554 (accessed Nov. 25, 2015).

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Antiviral activity of approved HCV direct-acting antivirals

Routine susceptibility In the absence of suitable cellular and animal models, cell-based culture systems for HCV (HCV replicons, HCV pseudo-particles [HCVpp], cell culture-derived HCV [HCVcc] and HCV trans-complemented JFH1 particles [HCVTCP]) have been developed (reviewed in (384, 385)). In concert with advances in determination of HCV protein structures, the advent of cell culture models have contributed to marked progress in the understanding of HCV-host cell interactions and viral and cellular factors contributing to viral replication. Both of these advances have allowed rapid development of HCV-specific DAAs.

The development of the HCV replicon system in 1999 significantly enhanced HCV drug development efforts (143). The HCV replicon system utilizes genetically engineered HCV ‘mini-genomes’ containing a selectable marker and an internal ribosome entry site which mediates translation of the HCV replicase (NS3 to NS5B). Following transfection into human hepatoma cell lines and marker selection, cell clones carrying high amounts of HCV RNA and proteins can be established. The disadvantage of the replicon system is that only the intracellular steps of the HCV replication cycle are replicated. Because most replication-enhancing mutations interfere with virus assembly (386), for a long time it was not possible to turn this system into a fully competent HCV cell culture model.

The second cell-based HCV culture system exploits pseudo-particle technology (HCVpp). Examples of its use include studying the neutralization of HCV by antibodies, the functionality of patient-derived HCV E1–E2 sequences, and the identification and characterization of HCV receptors (reviewed in (387)). The disadvantage of the HCVpp system is that only the very early stages of the HCV replication cycle are repeated.

Cell culture-derived HCV (HCVcc) replicates the complete viral replication cycle in cultured human hepatoma cells. This was possible with the identification of a particular HCV genotype 2a isolate cloned from a Japanese patient with fulminant hepatitis (isolate JFH-1) (388). Compared with other HCV isolates, the JFH-1 strain replicates to exceptionally high levels in the absence of replication-enhancing mutations. In vitro transcripts from a cloned JFH-1 genome can be transfected into human hepatoma cells, with resultant production of infectious (cell culture and in vivo) HCV particles. Chimeric HCV genomes have been developed for all seven HCV genotypes, encoding the structural proteins of HCV genotypes 1 to 7 and the non- structural proteins of JFH-1 with adaptive mutations (389). Subsequently, full-length cell

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culture-adapted HCV isolates derived from genotypes 1a, 2a and 2b have been developed that support the complete viral replication cycle in cultured human hepatoma cells (390, 391).

An alternative system to study the whole life cycle of HCV are trans-complemented JFH-1 particles (HCVTCP), generated using a JFH-1 sub-genomic replicon and an expression system for HCV proteins (core, E1, E2, p7, NS2) (392). Theoretically, the trans-complemented HCV can be derived from any patient isolate, providing isolate-specific information for HCV entry, replication and assembly.

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Supplementary Appendix Table 1-2. Antiviral activity of approved HCV protease inhibitors Median HCV HCV No of Compound Replicon EC Range genotype isolates 50 (nM) NS3/4A Protease Inhibitors First generation Telaprevir (VX-950) 1a 1 280 1b 1 354 Boceprevir (SCH-503034) 1b 1 200 Simeprevir (TMC-435) 1b 1 9.4 Paritaprevir (ABT-450) 1a 11 0.68 0.43-1.87 1b 9 0.06 0.03-0.09 2a 1 5.3 3a 1 19 4a 1 0.09 6a 1 0.68 Asunaprevir (BMS-650032) 1a - 4 1b - 1.2 2a - 230 2b - 480 3a - 1162 4a - 1.8 a Vaniprevir (MK-7009) 1b 1 3 2a 1 9 Second generation Grazoprevir (MK-5172) 1a 1 2a 1b 1 0.5a 2a 1 8a 3 1 13a a Mean EC50

Abbreviations: EC50, 50% effective concentration

Compiled from: (191, 205, 210, 211, 217, 224, 351, 393-410)

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Supplementary Appendix Table 1-3. Antiviral activity of approved HCV polymerase inhibitors Median HCV HCV No of Compound Replicon EC Range genotype isolates 50 (nM) NS5B RNA-dependent RNA Polymerase Inhibitors Nucleotide analogue Sofosbuvir (GS-7977) 1-6 14-110 1a 67 62 29-128 1b 29 102 45-170 2 15 29 14-81 3 106 81 24-181 Non-nucleoside inhibitor Dasabuvir (ABT-333) 1a 11 0.6 0.4-2.1 1b 10 0.3 0.2-2 a Mean EC50

Abbreviations: EC50, 50% effective concentration

Compiled from: (191, 205, 210, 211, 217, 224, 351, 393-410)

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Supplementary Appendix Table 1-4. Antiviral activity of approved HCV assembly inhibitors Median HCV HCV No of Compound Replicon EC Range genotype isolates 50 (nM) NS5A Inhibitors First generation, first wave Daclatasvir (BMS-790052) 1a 40 0.008 0.002-2409 1b 42 0.002 0.0007-10 2 16 16 0.005-60 3a 17 0.2 0.006-3.2 4 14 0.025 0.001-158 5 3 0.004 0.003-0.019 6 1 0.054 Ledipasvir (GS-5885) 1a - 0.018 0.009-0.085 1b - 0.006 0.004-0.007 2a - 21-249 2b 16-530 3a 1 168 4a 1 0.39 5a 1 0.15 6a 1 1.1 6e 1 264 Ombitasvir (ABT-267) 1a - 0.68 0.014-0.88 1b - 0.94 0.05-1.5 2a - 0.012 a 0.0082-0.012 2b - 0.0043 a 3a - 0.019 a 4a - 0.0017 a 5a - 0.0032 a 0.0032-0.0043 6a - 0.366 0.366-0.415 First generation, second wave Elbasvir (MK-8742) 1a 5 0.007 a 0.004-0.007 1b 4 0.003 a 2a 1 0.003 2b - 3.4 a 3a 1 0.14 a 0.03-0.14 4a - 0.003 Velpatasvir (GS-5816) 1-6 - 0.006-0.130 1a - 0.014 1b - 0.016 2a - 0.016 0.008-0.016 2b - 0.006 0.002-0.006 3a - 0.004 4a - 0.009 a Mean EC50

Abbreviations: EC50, 50% effective concentration Compiled from: (191, 205, 210, 211, 217, 224, 351, 393-410)

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Emerging resistance and cross-resistance Genotypic resistance analysis is based on DNA sequencing technologies. Population sequencing of the HCV genome detects viral variants at a frequency of approximately 20% within the HCV quasispecies while clonal and deep sequencing technologies allow detection of viral variants at a frequency of 0.5 - 1% (411). It remains unclear as to which frequency cut-off is most clinically relevant for prediction of virological failure.

Two concepts are important for understanding the virological and clinical significant of resistance. Firstly, the genetic barrier to resistance refers to the number and type of nucleotide changes (or substitutions) required for a virus to acquire resistance to an HCV DAA (412). Secondly, viral fitness refers to the relative capacity of a viral variant to replicate in a given environment (413).

Throughout this chapter, the nucleotide substitutions that confer resistance or reduced susceptibility to a drug or drug class will be referred to as “resistance-associated substitutions”, in preference to the term “resistance-associated variant “or “RAV” (414). Viral variants which harbour these resistance-associated substitutions and have reduced susceptibility to HCV DAAs will be referred to as “resistance variants” (414).

Prevalence of Resistance-Associated Substitutions in Direct-Acting Antiviral Naïve Individuals Resistance-associated substitutions may be present prior to treatment and can be selected for during treatment. Many viral variants harbouring these resistance-associated substitutions are unfit and may be undetectable prior to therapy, particularly on population-based sequencing. The natural prevalence of HCV resistance-associated substitutions has been assessed in a number of studies of treatment-naïve individuals, both by population (415-417) and next- generation sequencing (418-421). The majority of these studies focused on HCV genotype 1 infection. As such, the prevalence of resistance-associated substitutions in non-genotype 1 infections remains poorly characterized.

Overall, amino acid substitutions associated with resistance to nucleotide RdRp inhibitors (i.e., sofosbuvir) are rarely observed in untreated HCV patients, even at low frequency in the quasispecies population, as determined by next generation sequencing (419, 422). High viral fitness costs conferred by these substitutions (for example, S282T) are thought to prevent their emergence in untreated populations (423). Pre-existence of resistance-associated substitutions related to the non-nucleoside RdRp inhibitor, dasabuvir, are uncommon, but more frequent

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(HCV genotype 1: 0.2% - 3.1%) (211, 424). Exceptions to this are the naturally occurring variants C316N (genotype 1b: 11 - 36%) and S556G (genotype 1a: 7 - 25%); both are associated with low level resistance to dasabuvir (211, 422, 424).

In contrast, a potential challenge to widespread use of NS3/4A protease inhibitors is the pre- existence of resistance-associated substitutions in treatment-naïve individuals (prevalence of single resistance-associated substitutions in HCV genotype 1: 0.1 – 3.5%) (424, 425). This is most notable for the polymorphism Q80K, which compromises the efficacy of simeprevir, asunaprevir and paritaprevir in HCV genotype 1a infection. Within genotype 1a infected populations, the prevalence of Q80K varies geographically with recent analyses of Phase II and III clinical trials reporting prevalence rates of 9% in South America, 19 - 45% in Europe and 48% in North America (426-428). Given the underlying prevalence and impact on treatment response rates (in cirrhotic HCV genotype 1a infection), EASL and American Association for the Study of Liver Disease 2015 guidelines recommend the use of direct sequence analysis prior to initiation of treatment, and suggest avoiding simeprevir-containing regimens in patients with Q80K polymorphism (91, 103).

For NS5A assembly inhibitors, most single resistance-associated substitutions occur at a rate of 0.3 – 3.5% in treatment naïve individuals with genotype 1 infection, using population-based sequencing (424), with two notable exceptions for HCV genotype 1b. L31M (which confers low to medium level resistance to daclatasvir and ledipasvir) was observed in 2.1 - 6.3% and Y93H (which confers medium to high level resistance to daclatasvir, ledipasvir and ombitasvir) was observed in 3.8 - 19.0% (191, 211, 351, 421, 425). In HCV genotype 1 (predominantly 1b), Y93H is significantly associated with the favourable single-nucleotide polymorphisms, rs12979860 and rs8099917 (formerly IL28B), known to predict interferon-based treatment success (421, 425, 429). The clinical implications of this association are unclear. Although data is limited, Y93H was demonstrated in 2 - 3% of treatment naïve individuals with HCV genotype 3, using population-based sequencing (429, 430).

While overall the clinical importance of pre-treatment NS5A assembly inhibitor resistance- associated substitutions may be limited, a greater impact on treatment efficacy may be noted in individuals with prior interferon-based treatment and cirrhosis (421, 431). However, even in these populations, the combination of sofosbuvir and velpatasvir was highly effective in HCV genotype 1-6 populations (228, 333), demonstrating that resistance-associated substitutions can be overcome with potent regimens that have a high genetic barrier to resistance. In the ASTRAL 1 and 3 trials, NS5A assembly inhibitor resistance-associated substitutions were detected prior to treatment in 42% and 16% of individuals (with virological outcome data) who

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received sofosbuvir/velpatasvir, respectively, by next generation sequencing (cut-off 1%). In ASTRAL 1 (HCV genotypes 1, 2, 4, 5, 6), there was no difference in efficacy between those with and without NS5A resistance-associated variants at baseline (SVR12 99%) (228). In ASTRAL 3 (HCV genotype 3), treatment efficacy was reduced in those with resistance- associated variants A30K, L31M, and Y93H (SVR12 88% versus 97%; SVR12 with Y93H 84%) (333).

Treatment Emergent Resistance Associated Variants Resistance to HCV antiviral drugs is driven by the positive selection of viral variants with reduced susceptibility conferred by resistance-associated substitutions at different positions in the NS3/4a protease, the NS5B RdRp and the NS5A assembly protein (414, 424, 432). Each compound or drug class displays a specific substitution profile that may be influenced by the genotype and/or subtype with a difference in the genetic barrier to resistance, both within and between classes. Cross-resistance between compounds in the same inhibitor class is of most concern for NS3/4a protease inhibitors and NS5A inhibitors.

On the basis of in vitro data, resistance-associated substitutions can be grouped into those resulting in low-to-moderate fold-change (FC) (i.e., 5–20-fold) in the median effective concentration (EC50) and those resulting in high fold-change (i.e., >50-fold). However, the clinical importance of these arbitrary cut-offs is unclear.

HCV Protease Inhibitor Resistance Clinical trials with boceprevir and telaprevir, along with next-generation HCV protease inhibitors, have shown that a low barrier to resistance and extensive cross-resistance with selection of resistance-associated substitutions occurs when these drugs are used, especially as monotherapy (432, 433). Monotherapy with telaprevir resulted in the selection of resistant viral variants within the first week (205).

Examples of treatment-emergent resistance-associated substitutions (with a greater than or equal to 2 FC in EC50) related to NS3/4a inhibitors in HCV genotype 1 isolates are shown in Table 1.5. In general, additive resistance-associated substitutions confer greater reduction in susceptibility. For example, substitutions V36A/M, T54A/S, R155K/T, A156S, R155T+D168N, and V36A+T54A conferred 3-to 25-fold reduced susceptibility to telaprevir, while A156V/T substitutions and the V36M/A+R155K/T and T54S/A+A156S/T double substitutions conferred greater than 62-fold reduced susceptibility (205, 433). Substitutions at NS3 positions Q80, S122, R155, and/or D168 that were associated with lower reductions in

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susceptibility to simeprevir when occurring alone, reduced susceptibility to simeprevir by more than 50-fold when present in combination (217). Fortunately, many resistance-associated substitutions related to NS3/4A protease inhibitors are associated with impaired replicative fitness by HCV, and as such these variants are rapidly replaced by wild-type virus after stopping NS3/4A protease inhibitor therapy (433, 434). An exception is the Q80K variant which retains wild-type fitness and a relative high probability of pre-existence at baseline.

Next-generation NS3/4A protease inhibitors aim to overcome the issues associated with resistance demonstrated by first-generation protease inhibitors. In vitro, the median antiviral activity (EC50) of grazoprevir was similar in wild-type and mutant replicon systems against several substitutions that confer resistance to other protease inhibitors (Q41R, F43S, R155K, V36M, T54A/S, D168Y) (399). However, grazoprevir activity was markedly reduced against the A156T (genotype 1b) (399) and D168A/E (genotype 1a) substitution replicons (239, 435).

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Supplementary Appendix Table 1-5. Treatment-emergent resistance-associated substitutions (with >2 fold-change in EC50) related to NS3/4A inhibitors in HCV genotype 1

Resistance- Fold change in EC50 HCV associated HCV antiviral drugs [compared to WT replicon] gene substitution GT 1a GT 1b A156F/G/S NS3 BOC, TVR, ASV, VAN 2-10 2-20 BOC, TVR, ASV, PTV, A156T/V NS3 2-30 20->200 SMV, GZR, VAN ASV, PTV, SMV, GZR, D168A NS3 20-100 20-100 VAN ASV, PTV, SMV, GZR, D168C/E NS3 2-100 2-100 VAN ASV, PTV, SMV, GZR, D168G/N NS3 2-20 2-20 VAN ASV, PTV, SMV, GZR, D168H/T/K NS3 20->100 20->100 VAN D168V/Y NS3 ASV, PTV, SMV, VAN 20->200 >100 D168Y NS3 GZR 4 F43S NS3 ASV, SMV, GZR 2-20 F43I/V NS3 SMV 20-100 F43L NS3 PTV 20

M175L NS3 BOC 2-20

Q80K NS3 ASV, PTV, SMV 2-50 2-50 Q80R NS3 ASV, PTV, SMV, GZR 2-20 2-20 TVR, BOC, SMV, ASV, R155K1 NS3 2-100 2-100 PTV, GZR, VAN R155G/T NS3 BOC, TVR, PTV, GZR 2-20 2-20 R155G NS3 GZR >20

R155I/M/S/W NS3 TVR, PTV 2-20 2-100 S122R NS3 ASV, SMV 2-20 2-20 T54A/S NS3 BOC, TVR, VAN 2-20 2-20 BOC, TVR, PTV, GZR3, V36A/C/G/L/M 1 NS3 2-20 2-20 VAN V55A/I NS3 BOC, TVR 2-20 2-203 V170A/V NS3 BOC, TVR 2 2-20 V158I NS3 BOC 2 2-20 Y56H NS3 PTV 2-20 V36M+R155K NS3 PTV, VAN 50-600 Q80K+R155K NS3 PTV 20 V36L+Q80K+R155S NS3 GZR 50 1 R155K in combination with V36 variants results in improved viral fitness 2 V36A confers 2 fold-change to GZR in genotype 1b; no significant change for GZR activity in genotype 1a or 1b against other variants at codon 36. 3 V55I confers 2 fold-change to BOC with genotype 1b Note: Resistant substitutions are denoted using single-letter codes for amino acids, with wild-type first, followed by the position, and then the substitution amino acid; e.g., “R155K” denotes a change from the wild-type amino acid arginine to the variant amino acid lysine at position 155 of the protease gene.

Abbreviations: ASV, asunaprevir; BOC, boceprevir; GT, genotype; GZR, grazoprevir; PTV, paritaprevir; SMV, simeprevir; TVR, telaprevir; VAN, vaniprevir; WT, wild-type

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HCV NS5B RNA-dependent RNA Polymerase Inhibitor Resistance The HCV NS5B RNA-dependent RNA polymerase (RdRp) has been a prime target for antiviral development, given its vital role in viral replication. HCV antiviral drugs that bind to and inhibit the RdRp fall into two categories: nucleotide analogues and non-nucleoside inhibitors.

Sofosbuvir, the only approved NS5B nucleotide analogue RdRp inhibitor, has pan-genotypic activity and a high barrier to resistance (422). Reduced susceptibility to sofosbuvir is largely associated with the NS5B substitution S282T (Table 1.6) (398). In mutant replicons of HCV genotypes 1-6, the S282T substitutions conferred 2- to 18-fold reduced susceptibility to sofosbuvir, but also reduced HCV replication capacity by 89% to 99% compared to the corresponding wild-type virus (210, 398), limiting the clinical significance of this resistant variant. In a pooled analysis of subjects who received sofosbuvir in nine phase II and III clinical trials, no significant antiviral resistance to sofosbuvir was detected either at baseline or in cases of virological relapse except in one individual who received monotherapy (423). One individual who relapsed four weeks after treatment following 12 weeks of sofosbuvir monotherapy in the phase II ELECTRON trial had the S282T substitutions (325). However, by 12 weeks after treatment, the substitutions was no longer detectable by next generation sequencing, consistent with the impaired replicative capacity of the variant strain (436). In a recent meta-analysis of several sofosbuvir trials, substitutions at position 316 were found to be associated with failure of response to sofosbuvir, particularly for patients infected with genotype 1b (437). These substitutions were proposed to occupy a large spatial area within the catalytic site, thereby interfering with sofosbuvir entry and binding. The same study also identified L159F and V321A with the lack of response to sofosbuvir treatment (437). However, in cell culture models, these substitutions had only a minor effect on the potency of sofosbuvir when examined individually. Of clinical importance, retreatment with sofosbuvir-containing regimens has been successful in sofosbuvir-experienced individuals (438, 439).

Dasabuvir is a non-nucleoside RdRp inhibitor approved for use in HCV genotype 1 and 4 infection in combination with ombitasvir and ritonavir-boosted paritaprevir (440). Following exposure to dasabuvir in HCV genotype 1a replicons single NS5B substitutions C316Y, M414I/T, E446K/Q, Y448C/H, A553T, G554S, S556G/R, and Y561H reduced dasabuvir antiviral activity by 8- to 1,472-fold while in genotype 1b replicons, single NS5B substitutions C316H/N/Y, S368T, N411S, M414I/T, Y448C/H, A553V, S556G and D559G reduced dasabuvir antiviral activity by 5- to 1,569-fold (211).

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Supplementary Appendix Table 1-6. Treatment-emergent resistance-associated substitutions (with >2 fold-change in EC50) related to NS5B nucleotide and non-nucleoside polymerase inhibitors in HCV genotype 1

HCV Fold change in EC50 Resistance-associated HCV gene antiviral [compared to WT replicon] substitution drug GT 1a GT 1b S282T NS5B SOF 2-20 2-20 L159F + L320F NS5B SOF 2-5 2-5 A553T NS5B DSV >100 A553V NS5B DSV - >100 C316H/Y NS5B DSV >100 >100 C316N NS5B DSV 2-20 D559G NS5B DSV >100 >100 E446K/Q NS5B DSV 2-100 M414T NS5B DSV 20-100 20-100 M414I NS5B DSV 2-20 N411S NS5B DSV 2–20 S368T NS5B DSV >100 S556G NS5B DSV 2-20 2-20 S556N NS5B DSV 20-100 S556R NS5B DSV >100 Y448C NS5B DSV >100 >100 Y448H NS5B DSV >100 20-100 Y561H NS5B DSV 20-100 Comprehensive analysis of all NS5B population sequences in the SOF development program identified 2 NS5B variants, L159F and V321A, which emerged at the time of virological failure in 6 and 5 GT3- infected patients, respectively, using population based sequencing. However, the FC in EC50 of SOF against these single variants was <2 for L159F in GT1a, GT1b, and GT3a and <2 for V321A in GT3a (423, 441).

Note: Resistant substitutions are denoted using single-letter codes for amino acids, with wild-type first, followed by the position, and then the substitution amino acid; e.g., “R155K” denotes a change from the wild-type amino acid arginine to the variant amino acid lysine at position 155 of the protease gene.

Abbreviations: DSV, dasabuvir; GT, genotype; SOF, sofosbuvir

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HCV NS5A Assembly Inhibitor Resistance

Despite potent antiviral activity, resistance to NS5A inhibitors may be of greatest clinical importance in the current era of HCV antiviral drug therapy. Substitutions that confer resistance to NS5A inhibitors pre-exist in HCV quasispecies populations in the absence of any previous drug exposure. These variants generally replicate at low levels and are thus undetectable by currently available commercial assays, but they can be selected rapidly in the presence of an NS5A inhibitor. Clinically significant resistance is usually associated with an escape pattern whereby viral replication returns to pre-treatment levels and the dominant virus harbours amino acid substitutions that confer high levels of drug resistance without impairing viral fitness. In order to combat this, very high drug levels may be required to suppress resistant viruses; this necessity may have implications for patient safety.

High level resistance has emerged following exposure to all NS5A assembly inhibitors available to date (Table 1.7, Table 1.8), including the “second generation” NS5A inhibitors despite improvements in the genetic barrier to resistance. Studies of ombitasvir with HCV genotype 1a replicons revealed markedly reduced ombitasvir antiviral activity with single NS5A substitutions, M28T/V, Q30E/R, L31V, H58D, and Y93C/H/L/N (FC 58- to 67,000) (211, 405, 406). Of these, M28T had fitness equal to wild-type virus (100%), while mutations in codon 93 had a low fitness (18–25%) and the mutation in codon 30 had an intermediate fitness (60%)

(405). For ledipasvir, marked reduction in susceptibility (FC in EC50 >1000) was associated with substitution Y93H in genotypes 1a and 1b and Q30E in genotype 1a (351). In general, combination resistance-associated substitutions in HCV genotype 1a or 1b replicons further reduced antiviral activity (for example, ombitasvir: L31M + Y93H: FC in EC50, 12323; L31F +

Y93H: FC in EC50, 10270) (Table 1.9) (405).

Long-term follow-up data are required in populations with virological failure following NS5A inhibitor therapy. Of 152 HCV genotype 3-infected individuals treated in the ALLY-3 trial with daclatasvir plus sofosbuvir, 17 experienced virological failure (442). Virus from all 17 individuals at the time of virological failure demonstrated the presence of one or more NS5A resistance-associated substitutions, with the most common substitution at failure being Y93H (15/17) (442). Y93H was observed at baseline in six and emerged in nine individuals (442). Unlike the reversal to wild-type virus largely seen with treatment-emergent substitutions in NS5B RdRp and NS3/4A proteases, NS5A resistance-associated substitutions have been found to persist for more than a year after treatment in some individuals (191, 443). Fortunately, given the degree of resistance conferred, the Y93H substitution has been associated with poor replication fitness in genotype 1 isolates (405, 407).

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Supplementary Appendix Table 1-7. Single treatment-emergent resistance-associated substitutions (with ≥2 fold-change in EC50) related to NS5A inhibitors in HCV genotype 1

Resistance- Fold change in EC50 HCV associated HCV Antiviral Drug [compared to WT replicon] gene substitution GT 1a GT 1b A92K/T NS5A LDV 2-100 >100 H58D NS5A DCV, LDV, OMV, EBR 5->100 K24G/N/R NS5A LDV 2-100 L28M NS5A OMV, EBR 2 L28T NS5A DCV, OMV - 10- >100 L31I NS5A LDV 2-100 L31F NS5A DCV, OMV 5->100

L31F NS5A EBR 50-100 10-20 L31M NS5A DCV, LDV, OMV, EBR, VEL 2->100 2- >100 a L31V NS5A DCV, LDV, OMV, EBR 50->100 2-100 M28A/G NS5A DCV, LDV, EBR 50->100 M28T NS5A DCV, LDV, OMV, EBR, VEL 10 - >1000 M28V NS5A OMV 20-100 P32L/S NS5A DCV, LDV >100 2-100 P58D NS5A LDV >100 Q30D NS5A EBR >500 Q30E NS5A DCV, LDV, OMV 100->10000 Q30H NS5A DCV, LDV, OMV, EBR, VEL 2- >100 Q30R NS5A DCV, LDV, OMV, EBR, VEL 2->100 Q30G/K NS5A LDV >100 Q30L/T NS5A LDV 2-100 R30H NS5A DCV 2-20

S38F NS5A LDV 2-100

Y93C NS5A DCV, LDV, OMV, EBR, VEL 10 - >1000 Y93F NS5A LDV 2-100 Y93H NS5A DCV, LDV, OMV, EBR, VEL 100->10000 10->1000 Y93N NS5A DCV, LDV, OMV 100->10000 Y93S NS5A OMV >1000 a Elbasvir does not show reduced susceptibility to L31M in genotype 1b isolates (fold change=1)

Note: Resistant substitutions are denoted using single-letter codes for amino acids, with wild-type first, followed by the position, and then the substitution amino acid; e.g., “R155K” denotes a change from the wild-type amino acid arginine to the variant amino acid lysine at position 155 of the protease gene.

Abbreviations: DCV, daclatasvir; EBR, elbasvir; GT, genotype; LDV, ledipasvir; OMV, ombitasvir; VEL, velpatasvir

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Supplementary Appendix Table 1-8. Single treatment-emergent resistance-associated substitutions (with ≥2 fold-change in EC50) related to NS5A inhibitors in HCV genotypes 2 - 6

Resistance- HCV Fold change in EC50 HCV associated Antiviral [compared to WT replicon] gene substitution Drug GT 2 GT 3a GT 4a GT 5a GT 6a A30K NS5A DCV, EBR 20-100 C92R NS5A DCV 20-100 F28S NS5A DCV, OMV >100 L28F/I NS5A OMV 50->100 20-100 L28V NS5A OMV 20-100 L30H NS5A DCV >100 L31F NS5A DCV, OMV 20-500 >100

L31F NS5A EBR >100 DCV, LDV, L31M NS5A OMV, EBR, >100 >100 >100 VEL DCV, LDV, L31V NS5A >100 >100 >100 20-100 OMV, EBR DCV, LDV, M28T NS5A OMV, EBR, >100 VEL P32L/S NS5A DCV, LDV >100 Q24H NS5A DCV 20-100 T24A NS5A OMV 2-100 T58A/N/S NS5A DCV, OMV 10->100 DCV, LDV, Y93H NS5A OMV, EBR, >100 >100 >100 VEL Y93R NS5A DCV >100 Note: Resistant substitutions are denoted using single-letter codes for amino acids, with wild-type first, followed by the position, and then the substitution amino acid; e.g., “R155K” denotes a change from the wild-type amino acid arginine to the variant amino acid lysine at position 155 of the protease gene.

Abbreviations: DCV, daclatasvir; EBR, elbasvir; GT, genotype; LDV, ledipasvir; OMV, ombitasvir; VEL, velpatasvir

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Supplementary Appendix Table 1-9. Dual treatment-emergent resistance-associated substitutions (with ≥2 fold-change in EC50) related to NS5A inhibitors in HCV genotypes 1 and 3*

Resistance- HCV Fold change in EC50 HCV associated Antiviral [compared to WT replicon] gene substitution Drug GT 1a GT 1b GT 3a A30K + L31M NS5A DCV >1000 L28M + Y93H NS5A OMV >100 L31F + Y93H NS5A OMV >10000 L31I/M + Y93H NS5A DCV, OMV 100->1000 L31M + T21I NS5A DCV >500 L31V + Y93H NS5A DCV, OMV 100->10000 L31V + H58P NS5A DCV >50000 M28A/T + Q30K/R NS5A DCV >100000 P58L + Y93H NS5A OMV >1000 Q30D + Y93H NS5A EBR >20000 Q30H + H58D NS5A DCV >100000 Q30L + Y93H/S NS5A OMV >100 R30Q + Y93H NS5A OMV >100 S62A/T + Y93H NS5A DCV >1000 *Insufficient data for HCV GT 2, 4, 5 and 6

Note: Resistant substitutions are denoted using single-letter codes for amino acids, with wild-type first, followed by the position, and then the substitution amino acid; e.g., “R155K” denotes a change from the wild-type amino acid arginine to the variant amino acid lysine at position 155 of the protease gene.

Abbreviations: DCV, daclatasvir; EBR, elbasvir; GT, genotype; OMV, ombitasvir

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Pharmacokinetics of approved HCV direct-acting antivirals

See Supplementary Appendix Tables 1.10, 1.11 and 1.12 for summary data of important pharmacokinetic properties, divided by drug class (NS3/4A protease inhibitors, Table 1.10; NS5B nucleotide/non-nucleoside polymerase inhibitors, Table 1.11; NS5A inhibitors, Table 1.12).

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Supplementary Appendix Table 1-10. Summary of key pharmacokinetic and pharmacodynamic parameters of NS3/4A protease inhibitors Boceprevir Telaprevir Simeprevir Asunaprevir Paritaprevir Grazoprevir Bioavailability 800 mg three times 750 mg three times Adults 150 mg daily 100 mg twice daily 150 mg daily 100 mg daily daily daily 41 Plasma t (hours) 3 - 4 9 – 11 17 - 23 5.5 31 1/2 (200mg daily) Cmax (ng/ml) 1723 3510 2096 575 262 165 AUC (ng•h/ml) 2020-5408 22300 23740-57469 1887 2220 1420 Change in AUC with No significant Increase No significant No significant Increase Increase food administration change (moderate-high fat) change change Inter-individual Not determined 27% 87% Not determined 166% Not determined variation Protein binding 75% 59 - 76% >99.9% >99% 97-98.6% Not determined Volume of distribution 722 252 Not determined 194 16.7 1250 (L/kg) Total clearance, Cl/F 161 32 Not determined 50 Not determined 114 (L/hr) Renal clearance 3% 1% <1% <1% - <1% Metabolism Aldo- Metabolised by ketoreductase, CYP3A CYP3A4 CYP3A CYP3A4, CYP3A5 CYP3A CYP3A4/5 CYP2C, CYP3A, Inducer of - - CYP3A - -- CYP1A Intestinal CYP3A, CYP1A2, CYP2D6, CYP2C8, CYP3A4 CYP3A, P-gp, CYP2A6/C8/C19/ OATP1B1/1B3/2B UGT1A1, Inhibitor of CYP3A4/5 (strong) (weak), UGT1A1, OATP1B1/B2 D6, P-gp, BRCP, 1, P-gp, OAT1/3, OATP1B3, BRCP BCRP (gut) OATP1B1/B3, OCT1 MRP2 P-gp, MRP2, OATP1B1, P-gp, BCRP, P-gp, Transported by P-gp P-gp OATP1B1/B3, OATP2B1 OATP1B1/B3 OATP1B1/B3 OATP2B1

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Excretion 79% Urine 1% <1% <1% 8.8% <1% (3% unchanged) 9% 82% 91% 84% 88% Faeces >90% (8% unchanged) (32% unchanged) (31% unchanged) (7.5% unchanged) (1.1% unchanged) R-diastereomer No major 9 minor No circulating Metabolites Inactive (30-fold less - metabolites metabolites metabolites active) Abbreviations: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide (OATP), uridinediphosphate- glucuronosyltransferase (UGT), multidrug resistance-associated protein (MRP)

Compiled from: (205, 211, 217, 352, 396, 399, 444-446)

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Supplementary Appendix Table 1-11. Summary of key pharmacokinetic and pharmacodynamic parameters of NS5B polymerase inhibitors Sofosbuvir Dasabuvir (GS-331007)* Bioavailability Adults 400 mg daily 250 mg twice daily 0.4 Plasma T (hours) 5.5-6 1/2 (27) 603 Cmax (ng/mL) 667 (1378) 828-1010 AUC (ng.h/mL) 3240 (6790-7200) Change in AUC with food No significant change Increase administration Inter-individual variation Not determined 62% 61-65% Protein binding >99.5% (minimal) Volume of distribution (Liters/kg) Not determined 396 Total clearance, Cl/F (Liters/hr) Not determined Not determined - Renal clearance Not determined Not determined Metabolism CatA CYP2C8 Metabolised by CES1 CYP3A Hint1 Inducer of - - Inhibitor of - UGT1A1 Sofosbuvir only: P-gp, P-gp Transported by BCRP BCRP Excretion 80% Urine 2% (78%) 94% Faeces 14% (26% unchanged) Metabolites GS-331007 - *Following oral administration, >90% of systemic drug exposure is as GS-331007, which is phosphorylated to the active triphosphate catabolite; GS-331007 is the primary analyte of interest for pharmacokinetic analyses

Abbreviations: Human cathepsin A (CatA), carboxylesterase 1 (CES1), histidine triad nucleotide-binding protein 1 (Hint1), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) Compiled from: (210, 211, 445)

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Supplementary Appendix Table 1-12. Summary of key pharmacokinetic and pharmacodynamic parameters of NS5A inhibitors Daclatasvir Ledipasvir Ombitasvir Elbasvir Velpatasvir Bioavailability Adults 60 mg daily 90 mg daily 25 mg daily 50 mg daily 100 mg daily Plasma t1/2 (hours) 12 - 15 47 21 - 25 24 15 Cmax (ng/ml) 1534 323 68 121 259 AUC (ng•h/ml) 14122 7290 1000 1920 2980 Change in AUC with No significant change No significant change Increase No significant change No significant change food administration Inter-individual 20 - 40% Not determined 30% Not determined Not determined variation Protein binding >99% >99.8% 99.9% >99% >99.5% Volume of distribution 47 Not determined 50.1 680 Not determined (Liter/kg) Total clearance, Cl/F Not determined Not determined Not determined 26 Not determined (Liters/hr) Renal clearance 6.6% 1% - <1% <1% Metabolism Slow oxidative Amide hydrolysis, then CYP2B6, CYP2C8, Metabolised by CYP3A4 CYP3A metabolism oxidative metabolism CYP3A4 Inducer of CYP3A4 (weak) - - - P-gp P-gp BCRP Inhibitor of OATP1B1 - UGT1A1 BCRP (gut) OATP1B1/B3, BCRP OATP2B1 P-gp P-gp P-gp P-gp Transported by P-gp CYP3A4 BCRP BCRP BCRP OATP OATP1B1, OATP1B3 Excretion Urine 6.6% 1% 2% <1% 0.4% 88% 86% 90% Faeces >90% 94% (53% unchanged) (70% unchanged) (89% unchanged) Abbreviations: P-glycoprotein (P-gp), breast cancer resistance protein (BCCP), organic anion-transporting polypeptide (OATP) Compiled from: (191, 211, 351, 352, 408, 410, 445, 447-452)

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NS3/4A protease inhibitors

Telaprevir

Bioavailability Maximum plasma concentrations (Cmax) after a single dose are generally achieved after 4-5 hours (tmax). Exposure to telaprevir is higher during co-administration of PEG-IFNα and ribavirin than after administration of telaprevir alone. Because systemic exposure (AUC) to telaprevir was increased by 237% when telaprevir was administered with a standard fat meal compared to fasting conditions, telaprevir should always be taken with a standard fat meal).

Drug distribution Telaprevir is approximately 59-76% bound to plasma proteins (primarily alpha 1-acid glycoprotein and albumin) in a concentration-dependent manner, decreasing with increasing concentrations of telaprevir.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. There is no pharmacokinetic data in individuals with moderate (Childs Pugh B) or severe (Childs Pugh C) hepatic impairment. Use is not recommended in this population. Renal Impairment. After administration of a single 750 mg dose in HCV-negative adults with creatinine clearance (CrCl) <30 mL/min, the AUC increased by 21%. No dosage adjustments are required for renal impairment. Telaprevir has not been studied in HCV-infected adults with CrCl<50 mL/min, with end-stage renal disease or undergoing hemodialysis.

Excretion Metabolism. Telaprevir is extensively metabolized in the liver, involving hydrolysis, oxidation, and reduction. Multiple metabolites were detected in faeces, plasma, and urine, none of which were clinically significant. CYP3A4 was the major isoform responsible for CYP-mediated telaprevir metabolism while aldo-ketoreductases and other reductases are responsible for the reduction of telaprevir. Non-CYP mediated pathways of metabolism likely play a major role after multiple dosing of telaprevir. Elimination. Following administration of a single oral dose of 750 mg 14C-telaprevir in healthy subjects, 90% of total radioactivity was recovered in faeces (82%; 31.9% unchanged, 18.8% R- diastereomer), urine (1%) and expired air (9%).

Drug interactions Telaprevir is a strong inhibitor of CYP3A and as such, is contraindicated when combined with drugs that are highly dependent on CYP3A for clearance and for which elevated plasma concentrations are associated with serious and/or life-threatening events

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(narrow therapeutic index). Telaprevir is contraindicated when combined with drugs that strongly induce CYP3A and thus may lead to lower exposure and loss of efficacy of telaprevir. See Supplementary Appendix Table 1.13.

Supplementary Appendix Table 1-13. Examples of medications contraindicated for co- administration with telaprevir

Concomitant Drug Class: Effect on Clinical Comments Drug name Concentration Alpha 1-adrenoreceptor antagonist: Potential for hypotension or cardiac Alfuzosin ↑ alfuzosin arrhythmia Antimycobacterial: ↓↓↓ telaprevir Co-administration is not recommended Rifampicin Ergot derivative: Dihydroergotamine Potential for acute ergot toxicity Ergonovine ↑ ergot derivative characterized by peripheral vasospasm or Ergotamine ischemia Methylergonovine GI motility agent: ↑ cisapride Potential for cardiac arrhythmias Cisapride

Herbal supplements: ↓ telaprevir Co-administration is not recommended St. John's wort

HMG-CoA reductase inhibitor: ↑ HMG CoA Lovastatin Potential for myopathy including reductase Simvastatin rhabdomyolysis inhibitor Potential for serious and/or life-threatening Neuroleptic: ↑ pimozide adverse reactions such as cardiac Pimozide arrhythmias Potential for PDE5 inhibitor-associated PDE5 inhibitor: [for PAH] adverse events, including visual Sildenafil ↑ PDE5 inhibitor abnormalities, hypotension, prolonged Tadalafil erection, and syncope Sedatives/hypnotics: ↑ Prolonged or increased sedation or Midazolam (oral) sedative/hypnotic respiratory depression Triazolam Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org.

Abbreviations: PAH, pulmonary arterial hypertension

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Boceprevir

Bioavailability Boceprevir is administered as an approximately equal mixture of two diastereomers (1:1), SCH-534128 and SCH-534129, which rapidly interconvert in plasma. The predominant diastereomer (2:1), SCH-534128, is pharmacologically active and the other diastereomer is inactive. Boceprevir has a median tmax of 2 hours with Cmax 557ng/ml following a single 400 mg oral dose. Steady state AUC and Cmax increased in a less-than-dose- proportional manner and individual exposures overlapped substantially at 800 mg and 1200 mg, suggesting diminished absorption at higher doses. Steady state is achieved after approximately one day of three times daily dosing. As food enhanced the exposure of boceprevir by up to 65%, relative to the fasting state, boceprevir should always be given with food, regardless of type or timing of the meal.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. Following a single dose of 400mg of boceprevir in non-HCV-infected adults, the mean AUC and Cmax of the active diastereomer of boceprevir (SCH534128) were 32% and 28% higher in moderate (Child Pugh Class B) hepatic impairment, respectively and 45% and 62% higher in severe (Child Pugh Class C) hepatic impairment, respectively, relative to individuals with normal hepatic function. No significant difference in exposure was noted in those with mild (Child Pugh Class A) hepatic impairment. Renal Impairment. In non-HCV-infected adults with end-stage renal disease (ESRD) requiring haemodialysis following a single 800 mg dose of boceprevir, the mean AUC was 10% lower relative to those with normal renal function. Haemodialysis removed less than 1% of the boceprevir dose. No dosage adjustment is required for patients with any degree of renal impairment.

Excretion Metabolism. Studies in vitro indicate that boceprevir undergoes metabolism through the aldo- ketoreductase-mediated pathway to ketone-reduced metabolites that are inactive against HCV. To a lesser extent, boceprevir also undergoes oxidative metabolism mediated by CYP3A4/5. Elimination. Boceprevir is primarily eliminated by the liver. Following a single 800 mg oral dose of 14C-boceprevir, approximately 79% (8% unchanged) and 9% (3% unchanged) of the dose was excreted in faeces and urine, respectively.

Drug interactions See Supplementary Appendix Table 1.14.

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Supplementary Appendix Table 1-14. Examples of medications contraindicated for co- administration with boceprevir Concomitant Drug Class: Effect on Clinical Comments Drug name Concentration Alpha 1-adrenoreceptor antagonist Potential for hypotension or cardiac Alfuzosin ↑ alfuzosin arrhythmia Anticonvulsants: Carbamazepine

Phenobarbital Phenytoin Antimycobacterials: ↓↓↓ boceprevir Co-administration is not recommended Rifampicin Ergot derivatives: Dihydroergotamine Potential for acute ergot toxicity Ergonovine ↑ ergot derivative characterized by peripheral vasospasm or Ergotamine ischemia Methylergonovine GI motility agent: ↑ cisapride Potential for cardiac arrhythmias Cisapride Herbal supplement: ↓ boceprevir Co-administration is not recommended St. John's wort HMG-CoA reductase inhibitors: ↑ HMG CoA Lovastatin Potential for myopathy including reductase Simvastatin rhabdomyolysis inhibitor Oral contraceptives:

Drospirenone Potential for serious and/or life-threatening Neuroleptic: ↑ pimozide adverse reactions such as cardiac Pimozide arrhythmias Potential for PDE5 inhibitor-associated PDE5 inhibitor [for PAH]: adverse events, including visual Sildenafil ↑ PDE5 inhibitor abnormalities, hypotension, prolonged Tadalafil erection, syncope Sedatives/hypnotics: ↑ Prolonged or increased sedation or Midazolam (oral) sedative/hypnotic respiratory depression Triazolam Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org.

Abbreviations: PAH, pulmonary arterial hypertension

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Simeprevir

Bioavailability Plasma Cmax and AUC increased more than dose-proportionally after multiple doses between 75 - 200 mg once daily, with accumulation occurring following repeated dosing. Steady-state was reached after 7 days of once daily dosing. Plasma exposure (AUC) of simeprevir in HCV-infected adults was 2-3 fold higher compared to that observed in HCV- uninfected adults. Plasma Cmax and AUC of simeprevir were similar during co-administration of simeprevir with PEG-IFN α and ribavirin compared with administration of simeprevir alone. As administrating simeprevir with food to healthy subjects increased the relative bioavailability (AUC) and delayed absorption, simeprevir should be administered with food, regardless of type.

Drug distribution Simeprevir is extensively bound to plasma proteins (>99.9%), primarily albumin and, to a lesser extent, alfa 1-acid glycoprotein.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. Simeprevir is primarily metabolized by the liver. Compared to HCV- uninfected adults with normal hepatic function, the mean steady-state AUC of simeprevir was 2.4-fold higher in HCV-uninfected adults with moderate hepatic impairment (Child-Pugh Class B) and 5.2-fold higher in HCV-uninfected adults with severe hepatic impairment (Child-Pugh Class C). Simeprevir is not recommended for patients with moderate (Child-Pugh Class B) or severe hepatic impairment (Child-Pugh Class C), with post marketing reports of hepatic decompensation, hepatic failure, and death in patients with advanced or decompensated cirrhosis receiving simeprevir combination therapy.

Excretion Metabolism. Simeprevir is metabolized in the liver. Simeprevir primarily undergoes oxidative metabolism by the hepatic CYP3A system. Following a single oral administration of 200 mg (1.3 times the recommended dosage) 14C-simeprevir to healthy adults, the majority of the radioactivity in plasma was accounted for by unchanged drug and a small part of the radioactivity in plasma was related to metabolites (none being major). Metabolites identified in faeces were formed via oxidation and by O-demethylation followed by oxidation. Elimination. Elimination of simeprevir occurs via biliary excretion. Following a single oral administration of 200 mg 14C-simeprevir to healthy adults, 91% of the total radioactivity was recovered in faeces.

Drug interactions Simeprevir is metabolized by the cytochrome P450 system, primarily CYP3A, and is a substrate for several drug transporters, including organic anion transporting

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polypeptides (OATPs). It is susceptible to metabolic drug-drug interactions with agents that are moderate or strong CYP3A inhibitors (e.g. ritonavir and erythromycin) or CYP3A inducers (e.g. rifampin and efavirenz). Co-administration of these drugs may increase or decrease plasma concentrations of simeprevir, respectively, and should be avoided. Clinical studies have shown that simeprevir is a mild inhibitor of CYP1A2 and intestinal CYP3A but does not inhibit hepatic CYP3A. The effects of simeprevir on these enzymes are of clinical relevance only for narrow- therapeutic-index drugs that are metabolized solely by these enzymes (e.g. oral midazolam). Simeprevir is a substrate and inhibitor of the transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and OATP1B1/3. Cyclosporine is an inhibitor of OATP1B1/3, BCRP and P-gp, and a mild inhibitor of CYP3A; cyclosporine causes a significant increase in simeprevir plasma concentrations, and co-administration is not recommended. Clinical studies have demonstrated increases in co-administered drug concentrations for drugs that are substrates of the OATP1B1/3, BRCP (e.g. rosuvastatin) and P-gp (e.g. digoxin) transporters; these drugs should be administered with dose titration and or/close monitoring.

Post-marketing, life-threatening bradyarrhythmias have been documented in individuals taking amiodarone in combination with sofosbuvir-containing regimens, including sofosbuvir + simeprevir (215, 216). The mechanism for this effect is unknown.

Asunaprevir

Bioavailability In HCV-infected adults, asunaprevir Cmax occurred at tmax between 1 and 4 hours following a dose. Asunaprevir Cmax, AUC and Cmin increased in an approximately dose proportional manner. Steady state was achieved after 7 days of twice-daily administration in healthy subjects. The absolute oral bioavailability of the asunaprevir soft capsule was 9.3%. In healthy subjects, administration of 100 mg asunaprevir soft capsule with a high-fat meal increased the rate of absorption relative to fasting conditions, but did not have a clinically meaningful effect on the overall bioavailability.

Drug distribution Protein binding of asunaprevir in HCV-infected adults was greater than 99% and was independent of dose.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic impairment. Asunaprevir is metabolized via the liver and clinically significant increases in asunaprevir concentration were seen in individuals with moderate (Child-Pugh Class B) and severe (Child-Pugh Class C) hepatic impairment, limiting its use in this population (224, 453,

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454). In comparison with healthy adults, mild hepatic impairment did not result in a meaningful alteration in asunaprevir exposure. Renal Impairment. The pharmacokinetic properties of asunaprevir, as one of three components of a fixed dose combination tablet, with asunaprevir, daclatasvir and an investigational non- nucleoside NS5B inhibitor, were studied after multiple-dose administration in non-HCV infected adults with normal renal function (CrCl ≥90 mL/min) and mild (CrCl 60 to <90 mL/min), moderate (CrCl 30 to <60 mL/min) and severe (CrCl <30 mL/min) renal impairment not on haemodialysis, and with end-stage renal disease (ESRD) on haemodialysis. Asunaprevir unbound Cmax was estimated to be 37%, 87% and 119% higher and the asunaprevir unbound AUC was estimated to be 41%, 99% and 137% higher for individuals with mild, moderate and severe renal impairment, respectively, compared with subjects with normal renal function.

Asunaprevir unbound Cmax and AUC decreased 2% and 6%, respectively, soon after haemodialysis compared to individuals with normal renal function. As such, for individuals with severe renal impairment (CrCl <30 mL/min) who are not receiving hemodialysis, the dosing interval is prolonged with the recommended dose being 100 mg daily (as compared with the standard dose of 100 mg twice daily).

Excretion Metabolism. In vitro studies demonstrate that asunaprevir undergoes oxidative metabolism primarily mediated by CYP3A. Excretion. Following single-dose oral administration of 14C-asunaprevir in healthy subjects, 84% of total radioactivity was recovered in faeces (primarily as metabolites, 7.5% unchanged) and less than 1% was recovered in the urine (primarily as metabolites). Metabolism was the major route of asunaprevir elimination. Nine minor metabolites were detected in human plasma. Both asunaprevir and its metabolites were detected in human bile. .

Drug interactions CYP3A is involved in the elimination of asunaprevir. Therefore, moderate or strong inducers of CYP3A may decrease the plasma levels of asunaprevir, and moderate or strong inhibitors of CYP3A may increase the plasma levels of asunaprevir. Asunaprevir is also a substrate of P-gp. OATP1B1 and OATP2B1 are involved in the liver distribution of asunaprevir. Therefore, strong inhibitors of OATP-mediated transport may increase the plasma concentrations of asunaprevir and may decrease its therapeutic effect by reducing distribution to the liver. Clinically relevant examples of OATP inhibitors include ritonavir (OATPB1/B3/A2), atazanavir (OATPB1/B3), cyclosporine A (OATP1B1/B3/A2, OATP2B1), rifampicin (OATP1B1/B3, OATP2B1), lovastatin (OATP1BI), pravastatin (OATP1BI) and simvastatin (OATP1BI) (455, 456).

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Grazoprevir

Bioavailability In HCV-uninfected adults receiving single doses of grazoprevir, tmax was 2 – 5 hours and plasma t½ was 15 – 34.4 hours. Cmax and mean AUC increased in a dose-proportional fashion up to 200 mg and in a greater-than-dose-proportional fashion at doses above 200 mg. In HCV-uninfected adults receiving 10 days of grazoprevir (100 – 1000 mg daily), steady state was achieved after 6 days with median tmax of 2.5 – 4 hours and plasma t½ of 20 hours. In a similar multi-dose study in adults with HCV genotype 1 or 3, tmax was 2 – 4 hours and plasma t½ was 22 – 33 hours, with similar pharmacokinetic parameters across genotype 1 and 3. Based upon population pharmacokinetic modeling in HCV-infected adults, geometric mean AUC0-24 was 1420 ng.hr/ml (90% CI 1400, 1530) and Cmax was 165 ng/ml (90% CI 161, 176) with tmax at 2 hours (range 0.5 – 3 hours) (352). Drug exposure was 1.2–2.1 fold higher in HCV-infected individuals as compared with healthy controls (444, 457). Concomitant food administration did not significantly affect exposure (AUC).

Clinically important pharmacokinetic and pharmacodynamic features Hepatic impairment. In comparison with health controls, in non-HCV infected adults with mild (Child-Pugh Class A) and moderate (Child-Pugh Class B) hepatic impairment, grazoprevir exposure was increased approximately 2-fold and 5-fold, respectively (458). Renal impairment. Haemodialysis does not significantly affect grazoprevir pharmacokinetics in adults with end stage renal disease (ESRD). The removal of grazoprevir (<0.5%) by hemodialysis is negligible (446). The very high plasma protein binding of grazoprevir is consistent with the small amounts of grazoprevir quantified in dialysate. Grazoprevir concentrations were higher in adults with severe renal impairment not on haemodialysis compared to matched healthy adults. (446) Co-formulated grazoprevir/elbasvir for 12 weeks is safe and effective in individuals with HCV genotype 1 infection and advanced stage 4–5 chronic kidney disease, including those with ESRD on hemodialysis (459).

Excretion Grazoprevir is primarily eliminated through hepatic pathways, with minimal renal clearance.

Drug interactions Grazoprevir is a substrate of CYP3A, p-gp and OATP1B1/B3 transporters. Co-administration with drugs that inhibit OATP1B1/B3 may result in a significant increase in the plasma concentrations of grazoprevir and as such are contra-indicated. Clinically relevant examples of OATPP1B1/B3 inhibitors include ritonavir, atazanavir, cyclosporine A, rifampicin and simvastatin (455, 456). Co-administration with moderate or strong inducers of CYP3A may result in a significant decrease in the plasma concentrations of grazoprevir; as such, co-

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administration with strong CYP3A inducers is contra-indicated (e.g., efavirenz) and co- administration with moderate CYP3A inducers is not recommended. See Supplementary Appendix Table 1.15 for drugs contradicted for co-administration with grazoprevir/elbasvir.

Supplementary Appendix Table 1-15. Significant drug interactions with grazoprevir/elbasvir

Concomitant Drug Class: Effect on Clinical Comment Drug Name Concentration

Due to CYP3A induction, marked decreases Anticonvulsants: in GZR/EBR plasma concentrations are Carbamazepine ↓↓↓ GZR/EBR expected. Phenytoin Co-administration is contraindicated.

Antimycobacterials: Due to CYP3A induction, marked decreases Rifabutin in GZR/EBR plasma concentrations are ↓↓↓ GZR/EBR Rifampin expected. Rifapentine Co-administration is contraindicated.

Due to CYP3A induction, marked decreases Herbal Supplements: in GZR/EBR plasma concentrations are ↓↓↓ GZR/EBR St. John’s wort expected. Co-administration is contraindicated.

Due to CYP3A induction, marked decreases HIV non-nucleoside reverse ↓↓↓ GZR in GZR (>80%) and EBR plasma transcriptase inhibitors: ↓EBR concentrations are expected. Efavirenz Co-administration is contraindicated. HIV protease inhibitors: Atazanavir May increase risk of ALT elevation due to Darunavir increase in GZR concentration related to ↑ GZR Lopinavir inhibition of OATP1B1/B3 Saquinavir Co-administration is contraindicated. Tipranavir May increase risk of ALT elevation due to Immunosuppressant: increase in GZR concentration related to ↑ GZR Cyclosporine inhibition of OATP1B1/B3 Co-administration is contraindicated. Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org.

Abbreviations: GZR, grazoprevir; EBR, elbasvir

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NS5B polymerase inhibitors

Sofosbuvir

Following oral administration of sofosbuvir, the majority (>90%) of systemic drug exposure is as the inactive renally-excreted metabolite, GS-331007, which is considered the primary analyte of interest for the purposes of pharmacokinetic analysis. Sofosbuvir accounts for only 4% of systemic drug exposure, while GS-461203, the active triphosphate nucleotide analogue, is not detected in plasma.

Bioavailability Following oral administration of sofosbuvir, sofosbuvir was absorbed with plasma Cmax at a tmax of 0.5-2 hours post-dose with elimination t½ 0.5-1.5 hours. The GS-

331007 Cmax occurred at a tmax between 2-4 hours post-dose with elimination t½ 7-12 hours. Based on population pharmacokinetic analysis in HCV-infected adults who were co- administered ribavirin (with or without PEG-IFN), geometric mean steady state AUC 0-24 was 969 ng.hr/ml for sofosbuvir and 6790 ng.hr/mL for GS-331007, respectively. Relative to fasting conditions, the administration of a single dose of sofosbuvir with a standardized high fat meal did not substantially affect the sofosbuvir or GS-331007 exposure.

Clinically important pharmacokinetic and pharmacodynamic features Renal Impairment. The pharmacokinetics of sofosbuvir were studied in HCV negative subjects with mild (eGFR 50-80 mL/min/1.73m2), moderate (eGFR 30-50 mL/min/1.73m2), severe renal impairment (eGFR <30 mL/min/1.73m2) and subjects with end stage renal disease (ESRD) requiring haemodialysis following a single 400 mg dose of sofosbuvir. Relative to subjects with normal renal function (eGFR >80 mL/min/1.73m2), the sofosbuvir AUC was 61%, 107% and 171% higher in mild, moderate and severe renal impairment, while the GS-331007 AUC was 55%, 88% and 451% higher, respectively. In adults with ESRD, relative to adults with normal renal function, sofosbuvir and GS-331007 AUC was 28% and 1280% higher when sofosbuvir was dosed 1 hour before haemodialysis compared with 60% and 2070% higher when sofosbuvir was dosed 1 hour after haemodialysis, respectively. A 4-hour haemodialysis session removed approximately 18% of administered dose. While no dose adjustment is required for patients with mild or moderate renal impairment, safety and efficacy have not been established in patients with severe renal impairment or ESRD.

Excretion Metabolism. Sofosbuvir is extensively metabolized in the liver to form the pharmacologically active nucleoside analogue triphosphate, GS-461203. The metabolic activation pathway involves sequential hydrolysis (by carboxylesterase 1, cathepsin A and histidine triad nucleotide

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binding protein) followed by di- (mediated by UMP-CMP kinase) and tri-phosphorylation (mediated by nucleoside diphosphate kinase) to form GS-461203. Intrahepatic dephosphorylation of the monophosphate results in the formation of the nucleoside metabolite GS-331007 that cannot be efficiently rephosphorylated and lacks anti-HCV activity in vitro. Elimination. Following a single 400 mg oral dose of 14C-sofosbuvir, mean total recovery of the dose was greater than 92%, consisting of approximately 80%, 14%, and 2.5% recovered in urine, faeces, and expired air, respectively. The majority of the sofosbuvir dose recovered in urine was GS-331007 (78%). Renal clearance is the major elimination pathway for GS-331007.

Drug interactions Drugs that are potent P-gp inducers in the intestine (e.g., rifampin, St. John’s wort) may significantly decrease sofosbuvir plasma concentrations and may lead to a reduced therapeutic effect. Co-administration with drugs that inhibit P-gp and/or BCRP may increase sofosbuvir plasma concentration without increasing GS-331007 plasma concentration; accordingly, sofosbuvir may be co-administered with P-gp and/or BCRP inhibitors. Sofosbuvir and GS-331007 are not inhibitors of P-gp and BCRP and thus are not expected to increase exposures of drugs that are substrates of these transporters. The intracellular metabolic activation pathway of sofosbuvir is mediated by generally low affinity and high capacity hydrolase and nucleotide phosphorylation pathways that are unlikely to be affected by concomitant drugs. See Supplementary Appendix Table 1.16 for potentially significant drug interactions with ledipasvir/sofosbuvir and Supplementary Appendix Table 1.17 for potentially significant drug interactions with sofosbuvir/velpatasvir.

Post-marketing, life-threatening bradyarrhythmias have been documented in individuals taking amiodarone in combination with sofosbuvir-containing regimens, including sofosbuvir + ledipasvir, sofosbuvir + simeprevir and sofosbuvir + daclatasvir (215, 216). The mechanism for this effect is unknown.

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NS5A inhibitors

Daclatasvir

Bioavailability In dose ranging studies in HCV-infected subjects, geometric mean (CV%) daclatasvir Cmax was 1534 (58) mg/ml and AUC0-24 was 14122 (70) ng.h/ml with tmax at 1-2 hours post-dose. The absolute bioavailability of the tablet formulation is 67%. A food effect was not observed with administration of daclatasvir 60 mg tablet after a low-fat, low-caloric meal compared with fasted conditions.

Clinically important pharmacokinetic and pharmacodynamic features Renal Impairment. The pharmacokinetics of daclatasvir following a single 60 mg oral dose was studied in non–HCV infected individuals with renal impairment. The predicted AUC of daclatasvir was estimated to be 26%, 60%, and 80% higher in individuals with CrCl values of 60, 30, and 15 mL/min, respectively, relative to individuals with normal renal function (CrCl ≥90 mL/min). Individuals with end-stage renal disease (ESRD) requiring haemodialysis had a 27% increase in daclatasvir AUC and a 20% increase in unbound AUC compared to individuals with normal renal function. Daclatasvir is highly bound to plasma proteins and is unlikely to be removed by dialysis. Hepatic Impairment. The pharmacokinetics of daclatasvir following a single 30 mg oral dose was studied in non–HCV infected adults with mild (Child-Pugh Class A), moderate (Child-Pugh Class B), and severe (Child-Pugh Class C) hepatic impairment compared to a corresponding matched control group. The Cmax and AUC of total daclatasvir (free and protein-bound drug) were lower by 46% and 43%, respectively, in Child-Pugh Class A; by 45% and 38%, respectively, in Child-Pugh Class B; and by 55% and 36%, respectively, in Child-Pugh Class C.

The Cmax and AUC of unbound daclatasvir were lower by 43% and 40%, respectively, in Child- Pugh Class A; by 14% and 2%, respectively, in Child-Pugh Class B; and by 33% and 5%, respectively, in Child-Pugh Class C. Consequently, no dose adjustment is required for hepatic impairment.

Excretion Metabolism. Daclatasvir is a substrate of CYP3A, with CYP3A4 being the primary isoform responsible for metabolism. Following single-dose oral administration of 25 mg 14C-daclatasvir in healthy subjects, the majority of radioactivity in plasma was predominately attributed to parent drug (≥97%).

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Elimination. Following single-dose oral administration of 25 mg 14C-daclatasvir in healthy subjects, 88% of total radioactivity was recovered in faeces (53% unchanged) and 6.6% of the dose was excreted in the urine (primarily unchanged).

Drug interactions Daclatasvir is a substrate of CYP3A. Moderate or strong inducers of CYP3A markedly decrease the plasma levels and therapeutic effect of daclatasvir; consequently co-administration of daclatasvir with strong CYP3A inducers (e.g., carbamazepine, phenytoin, rifampin, St. John’s wort) is contraindicated. The dose of daclatasvir should be increased to 90 mg once daily when co-administered with moderate CYP3A inducers (e.g., efavirenz, etravirine, modafinil, nafcillin). Strong inhibitors of CYP3A (e.g., clarithromycin, azole antifungals, ritonavir) increase the plasma levels of daclatasvir. As such, when co-administered the dose of daclatasvir should be reduced to 30 mg once daily. Daclatasvir is an inhibitor of P-gp, OATP 1B1 and 1B3, and BCRP. Daclatasvir may increase systemic exposure to drugs that are substrates of P-gp, OATP 1B1 or 1B3, or BCRP, which could increase or prolong their therapeutic effect or adverse reactions.

Ledipasvir

Bioavailability Following oral administration of ledipasvir/sofosbuvir in healthy subjects, ledipasvir median Cmax were observed at tmax of 4 -4.5 hours post-dose. Relative to healthy subjects, ledipasvir AUC and Cmax were lower (24% and 32%, respectively) in HCV-infected subjects. Relative to fasting conditions, food with did not significantly effect sofosbuvir, GS- 331007 and ledipasvir exposure.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. Ledipasvir plasma exposure (AUC) after a single 90mg dose was similar in HCV-negative adults with severe hepatic impairment (Child-Pugh Class C) as compared with adults with normal hepatic function. Population pharmacokinetics analysis in HCV-infected subjects indicated that cirrhosis had no clinically relevant effect on the exposure of ledipasvir. Renal Impairment. No clinically relevant differences in ledipasvir pharmacokinetics were observed between healthy adults and adults with severe renal impairment (eGFR <30 mL/min).

Excretion Metabolism. Evidence of slow oxidative metabolism via an unknown mechanism has been observed. Following a single dose of 90 mg 14C-ledipasvir, systemic exposure was almost exclusively to the parent drug (>98%). Unchanged ledipasvir is the major species present in faeces.

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Elimination. Following a single 90 mg oral dose of 14C-ledipasvir, mean total recovery of the 14C in faeces and urine was approximately 87% (faeces 86%). Unchanged ledipasvir excreted in faeces accounted for a mean of 70% of the administered dose and the oxidative metabolite M19 accounted for 2.2% of the dose. Biliary excretion of unchanged ledipasvir is a major route of elimination with renal excretion being a minor pathway (1%).

Drug interactions Ledipasvir and sofosbuvir are substrates of drug transporters P-gp and BCRP while GS-331007 is not. P-gp inducers (e.g., rifampicin, St. John’s wort) may decrease ledipasvir and sofosbuvir plasma concentrations, leading to reduced therapeutic effect. Co- administration with drugs that inhibit P-gp and/or BCRP may increase ledipasvir and sofosbuvir plasma concentrations without increasing GS-331007 plasma concentration. See Supplementary Appendix Table 1.16.

Post-marketing, life-threatening bradyarrhythmias have been documented in individuals taking amiodarone in combination with sofosbuvir-containing regimens, including sofosbuvir/ledipasvir (215, 216). The mechanism for this effect is unknown.

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Supplementary Appendix Table 1-16. Potentially significant drug interactions with ledipasvir/sofosbuvir Concomitant Drug Class: Effect on Clinical Comment Drug Name Concentration Acid Reducing Agents: Antacids (e.g., aluminum Separate antacid and LDV/SOF by 4 hours. and magnesium hydroxide) Administer simultaneously with or 12 hours H2-receptor antagonists ↓ LDV apart from LDV/SOF at a dose that does not (e.g., famotidine) exceed famotidine 40 mg twice daily. Administer simultaneously with LDV/SOF Proton-pump inhibitors under fasted conditions at a dose comparable (e.g., omeprazole) to omeprazole 20 mg or lower Antiarrhythmic: Digoxin ↑ digoxin Consider therapeutic concentration monitoring Serious symptomatic bradycardia may occur in Effect on patients taking amiodarone, particularly if also amiodarone, receiving beta blockers, or those with Amiodarone LDV, SOF underlying cardiac comorbidities and/or concentrations advanced liver disease. Co-administration is unknown contraindicated. Anticonvulsants: Carbamazepine ↓ LDV Phenytoin ↓ SOF Co-administration is not recommended. Phenobarbital ↓ GS-331007 Oxcarbazepine Antimycobacterials: ↓↓ LDV Rifabutin ↓ SOF Co-administration is not recommended. Rifampin ↓ Rifapentine GS-331007 HIV Antiretroviral: Tenofovir disoproxil Clinical implications of an increase in fumarate (TDF) tenofovir concentration in this setting are + unclear. Nephrotoxicity has not been seen in Efavirenz ↑ tenofovir phase III clinical trials and real-world cohort HIV protease analyses. Monitor renal function. Tenofovir inhibitor/ritonavir alafenamide (TAF) may be an alternative. Elvitegravir/cobicistat ↓ LDV Tipranavir/ritonavir ↓ SOF Co-administration is not recommended. ↓ GS-331007 HCV Products: ↑ LDV Co-administration is not recommended. Simeprevir ↓ LDV Herbal Supplements: ↓ SOF Co-administration is not recommended St. John’s wort ↓ GS-331007 HMG-CoA Reductase Inhibitors: ↑ rosuvastatin Co-administration is not recommended. Rosuvastatin Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org.

Abbreviations: LDV, ledipasvir; SOF, sofosbuvir

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Elbasvir

Bioavailability Based upon population pharmacokinetic modelling in HCV-infected adults, geometric mean

AUC0-24 and Cmax were 1920 ng.hr/ml (90% CI 1880, 1960) and 121 (90% CI 118, 123), respectively with tmax occurring at 3 hours (range 3-6 hours) (352).

Clinically important pharmacokinetic and pharmacodynamic features Hepatic impairment. In HCV-uninfected adults with mild (Child-Pugh Class A) or moderate (Child-Pugh Class B) hepatic impairment, single 50 mg oral doses of elbasvir resulted in reductions in AUC (24%) and Cmax (42%) in mild hepatic impairment and reductions in AUC

(14%) and Cmax (31%) in moderate hepatic impairment compared with healthy controls (460). These results support the administration of elbasvir to adults with mild and moderate hepatic dysfunction without dose alteration. Renal impairment. Haemodialysis does not significantly affect elbasvir pharmacokinetics in adults with end stage renal disease (ESRD). The removal of elbasvir (0%) by hemodialysis is negligible (446). The very high plasma protein binding of elbasvir is consistent with undetectable levels of elbasvir in dialysate. Elbasvir concentrations were higher in adults with severe renal impairment not on haemodialysis compared to matched healthy adults. (446) Co- formulated grazoprevir/elbasvir for 12 weeks is safe and effective in individuals with HCV genotype 1 infection and advanced stage 4–5 chronic kidney disease, including those with ESRD on hemodialysis (459).

Excretion Elbasvir is primarily eliminated through hepatic pathways, with minimal renal clearance. .

Drug interactions Elbasvir AUC increased upon co-administration with ketoconazole (strong CYP3A4 and P-gp inhibitor), indicating that elbasvir is a substrate of CYP3A4 (451). See Supplementary Appendix Table 1.15 for drugs contraindicated with grazoprevir/elbasvir.

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Velpatasvir

Bioavailability Velpatasvir was absorbed rapidly with maximum plasma concentrations between 3 hours post dose (Cmax 259 ng/ml at tmax 3 hours) (410, 450). Plasma pharmacokinetics were similar across HCV genotypes 1 – 4 (410). Co-administration with food did not alter exposure to velpatasvir.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. Velpatasvir plasma exposure (AUC) was similar in individuals with moderate (Child-Pugh Class B) and severe (Child-Pugh Class C) hepatic impairment as compared with healthy controls. Renal Impairment. No clinically relevant differences in velpatasvir pharmacokinetics were observed between healthy subjects and subjects with severe renal impairment.

Drug interactions Velpatasvir is a substrate of P-gp, BCRP, OATP1B1 and OATP1B3. Velpatasvir is slowly metabolized by CYP2B6, CYP2C8 and CYP3A4 (353, 354, 449, 452, 461). Inducers of P-gp and moderate or potent inducers of BRCP and CYP2B6, CYP2C8, or CYP3A4 (e.g., rifampin, St. John’s wort, carbamazepine) should not be co-administered with sofosbuvir/velpatasvir, given the potential for reduced sofosbuvir/velpatasvir plasma concentration. Co-administration with rifampicin (potent P-gp and CYP inducer) reduced velpatasvir exposure by 70-80% (452). Co-administration with efavirenz (potent CYP2B6 and 3A4 inducer) reduced velpatasvir exposure by 50% (354). Velpatasvir is a weak inhibitor of P-gp and OATP1B and a moderate inhibitor of BRCP (449). See Supplementary Appendix Table 1.17.

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Supplementary Appendix Table 1-17. Significant drug interactions with sofosbuvir/velpatasvir Concomitant Drug Class: Effect on Clinical Comment Drug Name Concentration Acid Reducing Agents: Co-administration is not recommended Antacids (e.g., aluminum Separate antacid and SOF/VEL by 4 hours. and magnesium hydroxide) Administer simultaneously with or 12 hours H2-receptor antagonists ↓ SOF/VEL apart at a dose that does not exceed famotidine (e.g., famotidine) 20mg (or equivalent). Proton-pump inhibitors Administer simultaneously in the fed state at (e.g., omeprazole) doses equivalent to omeprazole 20 mg. Antiarrhythmic: Consider therapeutic concentration monitoring. Digoxin ↑ digoxin Co-administration is not recommended Serious symptomatic bradycardia may occur, particularly if also receiving beta blockers, or if Amiodarone - underlying cardiac comorbidities and/or advanced liver disease. Co-administration is contraindicated. Anticonvulsants: Carbamazepine Phenytoin ↓ SOF/VEL Co-administration is contraindicated. Phenobarbital Oxcarbazepine Antimycobacterials: Rifabutin ↓↓ SOF/VEL Co-administration is contraindicated. Rifampin Rifapentine Cardiac medications: Diltiazem Verapamil ↑ SOF/VEL Dronedarone or Co-administration is not recommended Quinine ↑ concomitant Bosentan medication Olmesartan Valsartan Herbal Supplements: St. John’s wort Echinacea ↓ SOF/VEL Co-administration is contraindicated. Milk thistle Chinese herb sho-saiko-to [VEL] ↓50% when co-administered with HIV NNRTI: efavirenz. While no data exists, similar effect is ↓↓ VEL Efavirenz likely with nevirapine and etravirine. Co- administration is contraindicated. Rosuvastatin ≤10mg daily. ↑ HMG CoA HMG-CoA Reductase Monitor for signs and symptoms of myopathy or reductase Inhibitors rhabdomyolysis. Co-administration is not inhibitor recommended. Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org.

Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; SOF, sofosbuvir; VEL, velpatasvir.

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Ombitasvir/paritaprevir/ritonavir and dasabuvir

Ritonavir has no direct antiviral action against HCV, but serves as a pharmacokinetic booster through its potent inhibition of cytochrome-P450. This in turn increases circulating concentrations of paritaprevir and decreases t½, allowing once-daily dosing.

Bioavailability Ombitasvir, paritaprevir, ritonavir and dasabuvir were absorbed after oral administration with mean tmax of 4 - 5 hours. While ombitasvir and dasabuvir exposures increased in a dose proportional manner, paritaprevir and ritonavir exposures increased in a more than dose proportional manner. Steady state exposures were achieved after approximately 12 days of dosing. The absolute bioavailability of dasabuvir was estimated to be approximately 70%. The absolute bioavailability of ombitasvir, paritaprevir, and ritonavir were not evaluated. Relative to fasting conditions, administration of ombitasvir, paritaprevir, ritonavir, and dasabuvir with a moderate or high fat meal increased the mean AUC, and as such, they should be administered with food with some fat.

Clinically important pharmacokinetic and pharmacodynamic features Hepatic Impairment. The single dose pharmacokinetics of ombitasvir, paritaprevir, ritonavir and dasabuvir were evaluated in non-HCV infected individuals with mild (Child-Pugh Class A), moderate (Child-Pugh Class B) and severe hepatic impairment (Child-Pugh Class C). Relative to individuals with normal hepatic function, ombitasvir, paritaprevir and ritonavir AUC values decreased by 8%, 29% and 34%, respectively, and dasabuvir AUC values increased by 17% in individuals with mild hepatic impairment. As such, no dosage adjustment is required for mild hepatic impairment (Child-Pugh Class A). Relative to individuals with normal hepatic function, ombitasvir, ritonavir and dasabuvir AUC values decreased by 30%, 30% and 16%, respectively, and paritaprevir AUC values increased by 62% in subjects with moderate hepatic impairment. This regimen is not recommended in HCV-infected individuals with moderate hepatic impairment (Child-Pugh Class B). Relative to individuals with normal hepatic function, paritaprevir, ritonavir and dasabuvir AUC values increased by 945%, 13%, and 325% respectively, and ombitasvir AUC values decreased by 54% in subjects with severe hepatic impairment. For these reasons, especially the enormous increase in ritonavir-boosted paritaprevir levels, this regimen is contraindicated in severe hepatic impairment (Child-Pugh Class C). Renal Impairment. The single dose pharmacokinetics of ombitasvir, paritaprevir, ritonavir and dasabuvir were evaluated in non-HCV infected individuals with mild (CrCl 60-89 mL/min), moderate (CrCl 30-59 mL/min), and severe (CrCl 15-29 mL/min) renal impairment. Overall, changes in exposure of ombitasvir, paritaprevir, ritonavir and dasabuvir in non-HCV infected

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individuals with mild-, moderate- and severe renal impairment are not expected to be clinically relevant.

Drug interactions Ombitasvir, paritaprevir, and dasabuvir are inhibitors of UGT1A1, and ritonavir is an inhibitor of CYP3A4. Paritaprevir is an inhibitor of OATP1B1 and OATP1B3 and paritaprevir, ritonavir and dasabuvir are inhibitors of BCRP. Co-administration of with drugs that are substrates of CYP3A, UGT1A1, BCRP, OATP1B1 or OATP1B3 may result in increased plasma concentrations of such drugs. Paritaprevir and ritonavir are primarily metabolized by CYP3A. Co-administration with strong inhibitors of CYP3A (e.g. clarithromycin, grapefruit juice, azole antifungals) may increase paritaprevir and ritonavir concentrations. Dasabuvir is primarily metabolized by CYP2C8 enzymes. Co-administration with drugs that inhibit CYP2C8 (e.g. gemfibrozil) may increase dasabuvir plasma concentrations. Ombitasvir is primarily metabolized via amide hydrolysis while CYP enzymes play a minor role in its metabolism. Ombitasvir, paritaprevir, dasabuvir and ritonavir are substrates of P-gp. Ombitasvir, paritaprevir and dasabuvir are substrates of BCRP. Paritaprevir is a substrate of OATP1B1 and OATP1B3. Inhibition of P-gp, BCRP, OATP1B1 or OATP1B3 may increase the plasma concentrations of the aforementioned DAAs. Clinically relevant examples of OATP inhibitors include ritonavir (OATPB1/B3/A2), atazanavir (OATPB1/B3), cyclosporine A (OATP1B1/B3/A2, OATP2B1), rifampicin (OATP1B1/B3, OATP2B1), lovastatin (OATP1BI), pravastatin (OATP1BI) and simvastatin (OATP1BI) (455, 456). See Supplementary Appendix Table 1.18 for contraindicated concomitant medications.

As ritonavir is also an HIV-1 protease inhibitor and can select for HIV-1 protease inhibitor resistance-associated substitutions, any HCV/HIV-1 co-infected individuals treated with ombitasvir/paritaprevir/ritonavir with or without dasabuvir should also be on a combination antiretroviral drug regimen with an HIV viral load <50 RNA copies/ml.

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Supplementary Appendix Table 1.18. Contraindicated concomitant medications with ombitasvir, paritaprevir and dasabuvir Concomitant Drug Class Drug name Effect on concentration Clinical Comments α1-adrenoreceptor antagonist Alfuzosin HCL Potential for hypotension. Carbamazepine Anticonvulsant Phenytoin ↓ PrOD Potential loss of anti-HCV therapeutic activity. Phenobarbital Antimycobacterial Rifampicin ↓ PrOD Potential loss of anti-HCV therapeutic activity. Dihydroergotamine Ergotamine Acute ergot toxicity (vasospasm, tissue ischemia) Ergot derivative ↑ concomitant medication Ergonovine associated with co-administration of ritonavir Methylergonovine Ethinyl estradiol-containing (ie, combined oral contraceptive) ALT elevation Herbal Supplement St. John’s Wort ↓ PrOD Potential loss of anti-HCV therapeutic activity. Lovastatin HMG-CoA Reductase Inhibitor ↑ concomitant medication Potential for myopathy including rhabdomyolysis. Simvastatin HIV integrase inhibitor with Elvitegravir/cobicistat ↑ PrOD pharmacokinetic booster Efavirenz HIV non-nucleoside reverse Etravirine ↑ liver enzymes transcriptase inhibitor Nevirapine Rilpivirine Potential for QTc prolongation. Indinavir ↑ in [P] due to CYP3A4 inhibition Lopinavir HIV protease inhibitor ↑ P Saquinavir *PrOD is a complete regimen containing ritonavir. Ritonavir* ↑ [D] by 10-fold – increases risk of QT Lipid-lowering agent Gemfibrozil ↑ D prolongation. Neuroleptic Pimozide Potential for cardiac arrhythmias. Sildenafil-associated adverse events such as visual PDE5 inhibitor (for PAH) Sildenafil ↑ concomitant medication disturbances, hypotension, priapism, and syncope. Triazolam Potential for prolonged or increased sedation or Sedatives/hypnotics ↑ concomitant medication Midazolam (oral) respiratory depression Note, this list is not exhaustive. Up to date drug-drug interaction information can be accessed via www.hep-druginteractions.org. Abbreviations: PAH, pulmonary arterial hypertension; PDE5, phosphodiesterase-5

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High adherence to short duration response-guided treatment among people with recent HCV infection: The ATAHC II and DARE-C I studies

Conference Presentation

Martinello M, Grebely J, Shaw D, Hellard M, Petoumenos K, Applegate T, Yeung B, Maire L, Iser D, Lloyd A, Sasadeusz J, Dore GJ, Matthews GV. High adherence to short duration response-guided treatment among people with recent HCV infection: The ATAHC II and DARE-C I studies [#112]. Poster session presented at: 4th International Symposium on Hepatitis Care in Substance Users; 2015 October 7-9; Sydney, NSW, Australia.

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Introduction: Adherence is associated with sustained virological response. This analysis assessed adherence and completion of response-guided pegylated interferon alfa-2a (PEG-IFN) and ribavirin among people with recent HCV infection.

Methods: Participants enrolled in the ATAHC II (PEG-IFN +/- ribavirin) and DARE-C I (PEG- IFN + ribavirin + telaprevir) studies were included. Treatment duration was response-guided (ATAHC II: 8, 16, 24 or 48 weeks; DARE-C I: 8, 12 or 24 weeks). PEG-IFN, ribavirin and telaprevir adherence were assessed by 80/80 (≥80% of doses, ≥80% treatment duration) and 100/100 adherence, on-treatment adherence and treatment completion. Logistic regression analyses were used to identify predictors of adherence. PEG-IFN 80/80 adherence and treatment completion were compared with ATAHC I (n=109, PEG-IFN +/- ribavirin for 24 weeks).

Results: 66 participants (94% male, 72% HIV; 59% had injected drugs ever) received treatment (PEG-IFN, n=1; PEG-IFN + ribavirin, n=51; PEG-IFN + ribavirin + telaprevir, n=14). Median treatment duration was 14 weeks (IQR 8-16). Early treatment discontinuation occurred in 18% (virological failure, 11% [n=7]; medical contraindication to continuation, 1% [n=1], clinician decision to cease, 5% [n=3]; participant unwilling to continue, 1% [n=1]), compared with 30% in ATAHC I (p=0.076). PEG-IFN 80/80 and 100/100 adherence were 100% and 98% (mean on- treatment adherence=99.9%). PEG-IFN 80/80 adherence was higher in ATAHC II/DARE-C I participants compared with ATAHC I (100% vs 82%, p<0.001). Ribavirin 80/80 and 100/100 adherence were 95% and 62% (mean on-treatment adherence=96.0%). RBV 80/80 adherence was higher in those with HCV/HIV (100% vs 82%, p=0.003). Ribavirin adherence was not associated with SVR12. In adjusted analysis, ribavirin adherence was associated with age (p=0.013), treatment regimen (p=0.012) and treatment duration (p=0.040), but not injection drug use.

Conclusion: Treatment adherence was high in participants receiving short duration response- guided therapy. Failure to achieve SVR was due to treatment discontinuation as opposed to non- adherence. Ribavirin adherence was associated with simpler treatment regimen and shorter treatment duration; this has implications for direct-acting antiviral therapy suggesting that reducing treatment duration and pill burden will benefit adherence.

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The management of recent HCV infection is not standardised with significant uncertainty regarding the optimal regimen and treatment duration, particularly as the therapeutic landscape changes with the advent of direct-acting antiviral (DAA) therapy (91, 103). Enhanced outcomes with interferon-based therapy in recent infection mean that treatment at this time can often be administered for a shorter duration (124, 128, 137, 138). Shorter treatment durations result in fewer adverse events, better quality of life, less frequent dose reductions and increased likelihood of optimal adherence (137).

With interferon-based therapy, adherence has been associated with sustained virological response (SVR) (462-465). However, studies of adherence in HCV therapeutics are limited by the restricted populations, small sample sizes and varying adherence definitions (464). Adherence refers to how closely an individual’s medication administration behaviour comes to the recommendations made by a provider with respect to timing, dosage and frequency and is often expressed as a percentage (466). Medication adherence research has been performed in a variety of chronic medical conditions, including diabetes, hypertension and HIV (467). Prior studies have demonstrated that on average, individuals consume 79% of prescribed medications doses (466). The term ‘non-adherence’ is often misused in regard to HCV infection and refers primarily to dose reductions by the clinician or early treatment discontinuation (466). In treatment of HCV infection, non-adherence is often defined as having received <80% of pegylated interferon (PEG-IFN) or ribavirin for <80% of the planned duration of therapy (462). In contrast, in the field of HIV, ‘non-adherence’ refers primarily to patient-missed doses (466). In an assessment of adherence to HCV therapy, it is important to consider non-adherence (missed doses) and treatment-discontinuations in assessing adherence (466).

While HCV treatment in PWID is feasible and successful across a broad range of multidisciplinary healthcare settings, treatment uptake remains low with multiple barriers to care at an individual and systems level, driven in part by concerns of adherence, social instability, treatment-related adverse effects, psychiatric comorbidity and the perceived risk of HCV reinfection (131-133, 137).

The aim of this analysis was to assess adherence and completion of short duration response- guided interferon-based therapy among people with recent HCV infection in the ATAHC II and DARE-C I studies.

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Methods

The study design, setting and participants for the Australian Trial in Acute Hepatitis C II (ATAHC II) and Direct-acting Antiviral Based Therapy for Recently Acquired Hepatitis C (DARE-C I) have been described in Chapter 2.

Briefly, ATAHC II was a prospective study of the natural history and treatment outcomes of recent HCV infection (estimated duration of infection ≤18 months) following response-guided therapy with PEG-IFN alfa-2a (180mcg/week) and ribavirin (genotype 1: 1000mg/day if <75kg, 1200mg/day if ≥75kg; genotype 2/3: 800mg/day). Treated participants received response- guided therapy with PEG-IFN with or without ribavirin, depending on duration of infection and HIV co-infection status (Supplementary Appendix Table 2-1). A sub-study of ATAHC II, DARE-C I assessed the efficacy and safety of response-guided therapy with PEG-IFN alfa-2a (180mcg/week), weight-based ribavirin (1000mg/day if <75kg, 1200mg/day if ≥75kg) and telaprevir (1125mg twice daily or 1125 three times daily if receiving efavirenz) for individuals with recent genotype 1 HCV infection (estimated duration of infection 6-18 months). Treatment duration was dependent on time to first HCV RNA below the limit of detection using COBAS Taqman HCV RNA assay (Supplementary Appendix Table 2-1).

Supplementary Appendix Table 2-1. Treatment allocation in ATAHC II and DARE C I HCV RNA Treatment duration Study Treatment regimen BLoD (weeks) Week 2 8 PEG-IFN +/- RBV Week 4 16 PEG-IFN +/- RBV ATAHC II Week 6 24 PEG-IFN +/- RBV Week 8 32 (24 for G2/3) PEG-IFN +/- RBV Week 12 48 (24 for G2/3) PEG-IFN +/- RBV Week 2 8 PEG-IFN/RBV/TVR

DARE-C I Week 4 12 PEG-IFN/RBV/TVR PEG-IFN/RBV/TVR for 12 weeks Week 8 24 + PEG-IFN/RBV for 12 weeks Abbreviations: BLoD, below limit of detection; PEG-IFN, pegylated interferon alfa-2a; RBV, ribavirin; TVR, telaprevir

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Study Definitions and Outcomes On-treatment adherence was calculated for each medication individually by subtracting the number of missed doses from the total number of doses prescribed for therapy duration and dividing by the total number of doses prescribed for therapy duration. By clinical assessment, pill count and self-reported questionnaire, compliance with each medication was individually calculated at the 80/80 and 100/100 adherence levels, defined as receipt of ≥80% or 100% of scheduled doses for ≥80% or 100% of the scheduled treatment period, respectively. PEG-IFN 80/80 adherence and treatment completion were compared with ATAHC I (n=109, PEG-IFN +/- ribavirin for 24 weeks).

Important definitions for analysis are listed below. • Non-adherence (missed doses): The number of missed doses during the total duration of treatment. • On-treatment adherence: Calculated by subtracting the number of missed doses from the total duration of treatment (week that treatment was discontinued or completed) and dividing by the total therapy duration. This measures the proportion of doses received from the time that treatment was initiated until treatment was discontinued or completed. • 80/80 PEG-IFN, ribavirin or telaprevir adherence: Defined as the receipt of ≥80% of scheduled PEG-IFN, ribavirin or telaprevir doses for ≥80% of the scheduled treatment period. For participants in whom therapy was terminated at 4-12 weeks due to virological non-response, the scheduled treatment period will be defined as 4-12 weeks. • 100/100 PEG-IFN, ribavirin or telaprevir adherence: Defined as the receipt of 100% of scheduled PEG-IFN, ribavirin or telaprevir doses for 100% of the scheduled treatment period. For participants in whom therapy was terminated at 4-12 weeks due to virological non-response, the scheduled treatment period will be defined as 4-12 weeks. • PEG-IFN or ribavirin dose-modification: A reduction in the dose of PEG-IFN or ribavirin at any time during treatment. • Early treatment discontinuation: Discontinuation of treatment prior to the per-protocol planned end of treatment. This includes participants with clinician-directed discontinuation for virological non-response. • Sustained virological response (SVR12): Defined as HCV RNA

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Study oversight All study participants provided written informed consent before study procedures. The study protocols were approved by St Vincent’s Hospital, Sydney Human Research Ethics Committee (primary study committee), as well as local ethics committees at all study sites. The studies were conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines. The studies were registered with clinicaltrials.gov registry (ATAHC II: NCT01336010; DARE C I: NCT01743521).

Statistical analysis

Among treated participants (n=66), non-adherence, on-treatment adherence, 80/80 and 100/100 adherence and early treatment discontinuation (number of weeks of therapy) were summarized using appropriate summary statistics.

Logistic regression analyses were used to identify predictors of 100/100 ribavirin adherence and early treatment discontinuation. Potential predictors were determined a priori and included sex, age, social functioning, injection drug use, HIV infection, HCV treatment regimen and HCV treatment duration. Social functioning was calculated using a validated scale from the Opiate Treatment Index (313) that addresses employment, residential stability, interpersonal conflict, social support, and the role of drug use in the participant’s social networks. A higher value indicates poorer functioning (range: 0– 48). The multivariate model was determined using a backwards stepwise approach, considering factors that were significant at the 0.2 level in univariate analysis. The final models included factors that remained significant at the 0.05 level. All p-values are two-sided. Analyses were performed using STATA version 14.0 (Stata Corporation, College Station, TX).

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Results

Supplementary Appendix Table 2-2. Baseline demographic and clinical characteristics, stratified by HIV co-infection Demographic and clinical Overall HCV HCV/HIV characteristics N=66 (%) N=18 (%) N=48 (%) Mean age (SD) 43 (11) 41 (12) 43 (10) Sex, n (%) Male 62 (94) 14 (78) 48 (100) Female 4 (6) 4 (22) 0 Caucasian, n (%) 58 (89) 15 (83) 43 (90) Mean weight (SD) 78.7 (13.1) 83.2 (18.7) 76.9 (10.1) Mean BMI (SD) 25.0 (3.3) 27.0 (4.4) 24.3 (2.5) Median social functioning score (IQR) 10 (6-15) 13 (9-20) 10 (6-14) Social functioning score, category <7 21 (32) 3 (17) 18 (38) 7-14 28 (42) 7 (39) 21 (44) >14 17 (26) 8 (44) 9 (19) Injection drug use, n (%) Ever 40 (61) 13 (72) 27 (56) Current a 28 (42) 10 (56) 18 (38) Opioid substitution treatment, n (%) Ever 8 (12) 4 (22) 4 (8) Current a 3 (5) 3 (17) 0 HCV genotype 1 42 (63) 10 (56) 32 (67) 2 1 (2) 1 (6) 0 3 22 (33) 7 (39) 15 (31) 4 1 (2) 0 1 (2)

Median log10 HCV RNA (IQR) 5.9 (5.1-6.6) 5.1 (4.9-5.8) 6.0 (5.3-6.7) HCV RNA >400,000 IU/mL 37 (56) 5 (28) 32 (67) Duration of infection at baseline 37 (30-46) (weeks), median (IQR) Treatment regimen PEG-IFN 1 (2) 1 (6) 0 PEG-IFN + RBV 51 (77) 14 (78) 37 (77) PEG-IFN + RBV + TVR 14 (21) 3 (17) 11 (23) Treatment duration (weeks), median 14 (8-16) 8 (8-16) 16 (8-20) (IQR) Treatment duration, category ≤8 weeks 25 (38) 10 (56) 15 (31) 9-16 weeks 26 (39) 5 (28) 21 (34) >16 weeks 15 (23) 3 (17) 12 (25) a Current refers to within 6 months of baseline visit (treatment day 1)

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Supplementary Appendix Table 2-3. Adherence to pegylated-interferon and ribavirin, stratified by HIV co-infection

Adherence Overall HCV HIV/HCV P Adherence to PEG-IFN N=66 N=18 N=48 Missed doses, n (%) No missed doses 65 (98) 17 (94) 48 (100) 1 missed dose 1 (2) 1 (6) 0 Mean on-treatment adherence, % (SD) 99.9 (0.2) 99.9 (0.5) 100 0.101 80% adherence, n (%) 66 (100) 18 (100) 48 (100) 100% adherence, n (%) 65 (98) 17 (94) 48 (100) PEG-IFN dose-modification 4 (6) 2 (11) 2 (4) 0.292

Adherence to ribavirin N=65* N=17* N=48 Missed doses, n (%) No missed doses 40 (62) 8 (47) 32 (67) 0.014 1-2 missed dose 15 (23) 3 (18) 12 (25) 3-10 missed doses 5 (8) 3 (18) 2 (4) 11-50 missed doses 2 (3) 1 (6) 1 (2) >50 missed doses 3 (5) 2 (12) 1 (2) Mean on-treatment adherence, % (SD) 96.0 (1.7) 86.3 (3.1) 99.5 (1.1) 0.004 80% adherence, n (%) 62 (95) 14 (82) 48 (100) 0.003 100% adherence, n (%) 40 (62) 8 (47) 33 (69) 0.153 Ribavirin dose-modification 6 (9) 1 (6) 5 (10) 0.579 *PEG-IFN monotherapy, n=1

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Supplementary Appendix Table 2-4. Factors associated with 100/100 ribavirin adherence – Logistic regression analysis <100% 100% OR P/ AOR P/ Variables adherence adherence (95% CI) P overall (95% CI) P overall N=25 (%) N=40 (%) Sex Male 23 (92) 38 (95) 1.00 - Female 2 (8) 2 (5) 0.63 (0.08, 4.60) 0.627 Age <45 19 (76) 20 (50) 1.00 - 1.00 - >45 6 (24) 20 (50) 3.11 (0.94, 11.61) 0.066 8.36 (1.58, 44.30) 0.013 Social functioning score 0.151 <7 4 (16) 15 (38) 1.00 - 7-14 13 (52) 18 (45) 0.37 (0.10, 1.37) 0.137 >14 8 (32) 7 (18) 0.23 (0.05, 1.04) 0.057 Injecting drug use – ever No 7 (28) 18 (45) 1.00 - Yes 18 (72) 22 (55) 0.48 (0.14, 1.55) 0.267 Injecting drug use – current 0.300 No 11 (44) 25 (63) 1.00 - Yes 13 (52) 13 (33) 0.44 (0.15, 1.25) 0.198 Unknown/missing 1 (4) 2 (5) 0.88 (0.07, 10.75) 0.920 Treatment regimen PEG-IFN and RBV 17 (68) 34 (85) 1.00 - 1.00 - PEG-IFN, RBV and telaprevir 8 (32) 6 (15) 0.38 (0.11, 1.26) 0.112 0.09 (0.02, 0.59) 0.012 Treatment duration, category 0.035 0.040 ≤8 weeks 9 (36) 16 (40) 1.0 - 1.00 - 9-16 weeks 6 (24) 19 (46) 1.78 (0.52, 6.09) 0.357 1.50 (0.37, 6.16) 0.570 >16 weeks 10 (40) 5 (13) 0.28 (0.07, 1.08) 0.065 0.20 (0.04, 0.95) 0.043 HIV infection No 9 (36) 8 (20) 1.00 - Yes 16 (64) 32 (80) 2.25 (0.73, 6.94) 0.158

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Supplementary Appendix Table 2-5. Factors associated with early treatment discontinuation (n=66) - Logistic regression

Early Treatment P/ treatment OR Variables completion P discontinuation (95% CI) N= 54 (%) overall N=12 (%) Sex Male 11 (92) 51 (94) 1.00 - Female 1 (8) 3 (6) 1.55 (0.15, 16.29) 0.717 Age (category) 1.16 (0.87, 1.57) 0.319 <45 6 (50) 33 (61) 1.00 - >45 6 (50) 21 (39) 1.56 (0.36, 6.72) 0.694 Higher education No 4 (33) 19 (35) 1.00 - Yes 8 (67) 35 (65) 1.09 (0.29, 4.08) 0.903 Social functioning score 0.950 <7 3 (25) 16 (30) 1.00 - 7-14 6 (50) 25 (46) 1.28 (0.28, 5.86) 0.750 >14 3 (25) 13 (24) 1.23 (0.21, 7.15) 0.817 Injecting drug use – ever No 7 (58) 19 (35) 1.00 - Yes 5 (42) 35 (65) 0.39 (0.11, 1.39) 0.146 Injecting drug use – current No 8 (67) 29 (54) 1.00 - Yes 4 (33) 22 (41) 0.66 (0.18, 2.47) 0.537 Unknown/missing 0 3 (6) 1.00 - HIV infection No 2 (17) 16 (30) 1.00 - Yes 10 (83) 38 (70) 2.11 (0.41, 10.71) 0.370 Treatment regimen PEG-IFN +/- RBV 10 (83) 42 (78) 1.00 - PEG-IFN, RBV + telaprevir 2 (17) 12 (22) 0.70 (0.13, 3.64) 0.671

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Supplementary Appendix Table 2-6. Impact of ribavirin adherence on SVR (n=65) – Univariate logistic regression analysis

SVR No SVR OR P/ Variables N=47 N=18 (95% CI) P overall Missed ribavirin doses, n (%) No missed doses 28 (60) 13 (72) 1.00 - Any missed doses 19 (40) 5 (28) 1.29 (0.68, 2.45) 0.431 On-treatment ribavirin adherence, (%) ≥80% adherence 45 (96) 17 (94) 1.00 - <80% adherence, n (%) 2 (4) 1 (6) 1.32 (0.11, 15.56) 0.824 On-treatment ribavirin adherence, (%) 100% adherence 28 (60) 13 (72) 1.00 - <100% adherence, n (%) 19 (40) 5 (28) 0.57 (0.17, 1.85) 0.348 On-treatment adherence, category 0.566 <80% 2 (4) 1 (6) 1.00 - 80-99% 17 (36) 4 (22) 2.12 (0.15, 29.66) 0.575 100% 28 (60) 13 (72) 1.08 (0.09, 12.98) 0.953

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Treatment Treatment period (weeks) duration PEG-IFN Ribavirin SVR 12 (weeks) 0-4 4-8 8-12 12-16 16-20 20-24 24-48 0-4 4-8 8-12 12-16 16-20 20-24 24-48 1 No 2 No

2 No

5 No

6 * No 8 * Yes

8 No

8 * Yes

8 * Yes

8 No

8 * Yes

8 * Yes

8 * Yes

8 * Yes

8 * Yes

8 * Yes

8 * Yes

8 * Yes

8 * Yes 8 * Yes 8 * Yes 8 No 8 * Yes 8 No 8 * Yes 12 No

12 No

12 No

12 No

12 No

12 * Yes 12 * Yes 12 * Yes 16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 * Yes

16 No

16 * Yes

16 * Yes

16 * Yes

24 * Yes

24 No

24 * Yes

24 * Yes

24 * Yes

24 * Yes

24 * Yes

24 No

24 * Yes

24 * Yes

24 No

24 * Yes 24 * Yes 48 * Yes

48 * Yes SVR12 indicated by * Figure 100% 90-99% 90-50% <50% No missed doses I missed dose legend dose dose dose dose

Supplementary Appendix Figure 2-1. On-treatment adherence in ATAHC II and DARE-C I

Abbreviations: SVR, sustained virological response

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List of investigators

ATAHC I Coordinating investigators: A/Prof Gail V Matthews, Prof Gregory J Dore

Clinical Site Principal Investigators: Dr David Baker, 407 Doctors, Sydney, NSW, Australia Dr Mark Bloch, Holdsworth House GP Practice, Sydney, NSW, Australia Dr Ingrid van Beek, Kirketon Road Centre, Sydney, NSW, Australia Dr Darrell Crawford, Princess Alexandra Hospital, Brisbane, QLD, Australia Dr Paul Desmond, St Vincent’s Hospital, Melbourne, VIC, Australia Prof Gregory Dore, St Vincent’s Hospital, Sydney, NSW, Australia Prof Jacob George, Westmead Hospital, Sydney, NSW, Australia Prof Paul Haber, Royal Prince Alfred Hospital, Sydney, NSW, Australia Prof Margaret Hellard, Infectious Disease Unit, The Alfred Hospital, Melbourne, VIC, Australia Dr Brian Hughes, John Hunter Hospital, Newcastle, NSW, Australia Dr Lindsay Mollison, Fremantle Hospital, Fremantle, WA, Australia Dr Nghi Phung, Nepean Hospital, Sydney, NSW, Australia Prof Stuart Roberts, Gastroenterology Unit, The Alfred Hospital, Melbourne, VIC, Australia A/Prof Joe Sasadeusz, Royal Melbourne Hospital, Melbourne, VIC, Australia A/Prof David Shaw, Royal Adelaide Hospital, Adelaide, SA, Australia Prof William Sievert, Monash Medical Centre, Melbourne, VIC, Australia

ATAHC II Coordinating investigators: A/Prof Gail V Matthews, Prof Gregory J Dore

Clinical Site Principal Investigators: Prof Paul Haber, Royal Prince Alfred Hospital, Sydney, NSW, Australia Prof Margaret Hellard, Infectious Disease Unit, Alfred Hospital, Melbourne, VIC, Australia A/Prof Gail V Matthews, St Vincent’s Hospital, Sydney, NSW, Australia Dr Phillip Read, Kirketon Road Centre, Sydney, NSW, Australia A/Prof Joe Sasadeusz, Royal Melbourne Hospital, Melbourne, VIC, Australia A/Prof David Shaw, Royal Adelaide Hospital, Adelaide, SA, Australia Dr Alexander Thompson, St Vincent’s Hospital, Melbourne, VIC, Australia

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DARE-C I Coordinating investigator: A/Prof Gail V Matthews

Clinical Site Principal Investigators: Prof Gregory J Dore, St Vincent’s Hospital, Sydney, NSW, Australia Prof Margaret Hellard, Infectious Disease Unit, Alfred Hospital, Melbourne, VIC, Australia A/Prof David Shaw, Royal Adelaide Hospital, Adelaide, SA, Australia

DARE-C II Coordinating investigator: A/Prof Gail V Matthews

Clinical Site Principal Investigators: Prof Gregory J Dore, St Vincent’s Hospital, Sydney, NSW, Australia Prof Edward Gane, Auckland Hospital, Auckland, New Zealand Prof Margaret Hellard, Infectious Disease Unit, Alfred Hospital, Melbourne, VIC, Australia A/Prof Joe Sasadeusz, Royal Melbourne Hospital, Melbourne, VIC, Australia A/Prof David Shaw, Royal Adelaide Hospital, Adelaide, SA, Australia

CEASE Coordinating investigator: A/Prof Gail V Matthews

Clinical Site Principal Investigators: Prof Gregory J Dore, St Vincent’s Hospital, Sydney, NSW, Australia Dr Rohan Bopage, The Albion Centre, Sydney, NSW, Australia Dr Robert Finlayson, Taylor Square Private Clinic, Sydney, NSW, Australia Dr David Baker, East Sydney Doctors, Sydney, NSW, Australia Dr Mark Bloch, Holdsworth House Medical Practice, Sydney, NSW, Australia

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