Agarwal, Kosh

Aldersley, Mark A.

Aranday-Cortes, Elihu

da Silva Filipe, Ana

Foster, Graham R

Hughes, Joseph

Irving, William L

Macdonald, Douglas

McLauchlan, John

Richardson, Paul

Rosenberg, William

Sreenu, Vattipally

Thomson, Emma C.

Ustianowski, Andrew

Wiselka, Martin

Journal of Hepatology

English

Ana da Silva Filipe

Vattipally Sreenu

Joseph Hughes

Elihu Aranday-Cortes

William L. Irving

Graham R. Foster

Kosh Agarwal

William Rosenberg

Douglas Macdonald

Paul Richardson

Mark A. Aldersley

Martin Wiselka

Andrew Ustianowski

John McLauchlan

Emma C. Thomson

Crossref

Response to DAA therapy in the NHS England Early Access Programme for rare HCV subtypes from low and middle income countries

King's Research Portal

                       King’s Research Portal  DOI:10.1016/j.jhep.2017.06.035Document VersionPeer reviewed versionLink to publication record in King's Research PortalCitation for published version (APA):da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W. L., Foster, G. R., Agarwal, K.,Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M. A., Wiselka, M., Ustianowski, A., McLauchlan, J.,& Thomson, E. C. (2017). Response to DAA Therapy in the NHS England Early Access Programme for RareHCV Subtypes from Low and Middle Income Countries. Journal of Hepatology. Advance online publication.https://doi.org/10.1016/j.jhep.2017.06.035Citing this paperPlease note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this maydiffer from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination,volume/issue, and date of publication details. 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Jan. 2026Accepted ManuscriptResponse to DAA Therapy in the NHS England Early Access Programme forRare HCV Subtypes from Low and Middle Income CountriesAna da Silva Filipe, Vattipally Sreenu, Joseph Hughes, Elihu Aranday-Cortes,William L Irving, Graham R. Foster, Kosh Agarwal, William Rosenberg,Douglas Macdonald, Paul Richardson, Mark A. Aldersley, Martin Wiselka,Andrew Ustianowski, John McLauchlan, Emma C. ThomsonPII: S0168-8278(17)32195-5DOI: http://dx.doi.org/10.1016/j.jhep.2017.06.035Reference: JHEPAT 6615To appear in: Journal of HepatologyReceived Date: 28 June 2017Accepted Date: 29 June 2017Please cite this article as: da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W.L., Foster, G.R.,Agarwal, K., Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M.A., Wiselka, M., Ustianowski, A.,McLauchlan, J., Thomson, E.C., Response to DAA Therapy in the NHS England Early Access Programme for RareHCV Subtypes from Low and Middle Income Countries, Journal of Hepatology (2017), doi: http://dx.doi.org/10.1016/j.jhep.2017.06.035This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  Response to DAA Therapy in the NHS England Early Access Programme for Rare HCV Subtypes from Low and Middle Income Countries   Ana da Silva Filipe1, Vattipally Sreenu1, Joseph Hughes1, Elihu Aranday-Cortes1, William L Irving2, Graham R. Foster3, Kosh Agarwal4, William Rosenberg5, Douglas Macdonald5, Paul Richardson6, Mark A. Aldersley7, Martin Wiselka8, Andrew Ustianowski9, John McLauchlan1, Emma C. Thomson1 1. MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.  2. Gastrointestinal and Liver Disorders Theme, NIHR Nottingham Biomedical Research Centre at the Nottingham University Hospitals NHS Trust and the University of Nottingham, UK. 3. Blizard Institute, Queen Mary University of London, London, UK.  4. Institute of Liver Studies, King's College Hospital Foundation Trust, London UK.  5. Institute of Liver and Digestive Health, University College of London, London, United Kingdom. 6. Royal Liverpool University NHS Trust, Liverpool, UK.  7. Liver Unit, St James’s University Hospital, Leeds, UK.  8. Infectious Diseases Unit, University Hospitals Leicester NHS Trust, Leicester, UK.  9. Infectious Diseases, North Manchester General Hospital, Manchester, UK.  Financial Support: This work was supported by grants from the Medical Research Foundation to HCV Research UK (C0365) and the UK Medical Research Council (MRC) to the STOP-HCV consortium (MR/K01532X/1). AS, VS, JH, EAC, JM and ECT were also supported by the MRC (MC.UU.12014/1) and ECT was funded by the Wellcome Trust (102789/Z/13/Z). We are also grateful to Gilead Sciences and Bristol Myers Squibb for financial support. Correspondence: Prof J McLauchlan, MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, Glasgow G61 1QH, UK; E-mail,  john.mclauchlan@glasgow.ac.uk; Tel no. +44 141 330 4028. Conflict of Interest: The authors disclose the following conflicts: WLI has received speaker and consultancy fees from Roche Products, Janssen Cilag and Novartis, educational grants   from Boehringer Ingelheim, MSD and Gilead Sciences, and research grant support from GlaxoSmithKline, Pfizer, Gilead Sciences and Janssen Cilag; GRF has received research grants from Roche, Gilead Sciences, Springbank, and received speaker and consultancy fees from AbbVie, Gilead Sciences, MSD, GSK and Janssen Pharmaceuticals; KA has received speaker and consultancy fees from AbbVie, Achillion, Astellas, BMS, Gilead Sciences, GSK, Janssen, MSD, Novartis; WR is among the inventors and patent holders for the ELF® Test and receives consultancy fees from Siemens; MAA has been an advisory board member and in receipt of educational grants from Gilead Sciences; AU has received speaker fees and advisory board fees from Gilead Sciences, ViiV, Abbvie, MSD, Janssen and BMS; JM has received speaker and consultancy fees from Abbvie, Gilead Sciences and BMS. The remaining authors have no conflicts to declare. Author Contributions: The study was designed and led by AS, JM and ECT. EAC, VS and JH formed NGS and host genetic analysis. WLI collated and provided the clinical data from HCV Research UK. GRF, KA, WR, DM, PR, MAA, MW and AU consented the patients, provided clinical data and provided samples. All authors contributed to and commented on the drafting of the final manuscript.   To the Editor:  We read with interest “NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome” by Zeuzem et al. in the Journal of Hepatology, which indicated lower SVR rates for Gt1a and Gt1b strains carrying multiple resistance-associated substitutions (RASs) in NS5A [1]. Their study is very comprehensive but, as the authors note, it is largely restricted to HCV subtypes Gt1a and Gt1b, and does not represent subtypes from large geographical regions, including Africa. Indeed, current guidelines for treatment with direct-acting antivirals (DAAs) [2] are based on trial data for a limited selection of HCV genotypes, typically found in high-income countries. However, HCV is highly variable with natural polymorphisms at sites of DAA resistance. Our analysis of patients originating from low- and middle-income countries (LMICs), recruited into the NHS England Early Access Programme (EAP), has identified HCV subtypes, which contain naturally-occurring single and double RAS in NS5A that are associated with apparent poor response to approved DAAs. Previously, we described clinical outcomes from the EAP cohort, which provided IFN-free, DAA therapy to >800 HCV-infected patients with decompensated liver disease [3, 4]; all patients received sofosbuvir±ribavirin (SOF±RBV) combined with either ledipasvir (LDV) or daclatasvir (DCV). Among the Gt1-infected group, 28 patients did not achieve a SVR. From next generation sequence (NGS) analysis, 3 of these patients, designated as Gt1- or Gt1a-infected by the clinical sites, were infected with Gt1l. All 3 patients originated from Nigeria. The patients were retreated with DAAs for a further 24 weeks but again relapsed after therapy. In two Gt1l- infected patients (P1 and P3) where pre- and post-treatment samples were available, there were no sequence changes in either NS5A or NS5B. By contrast, NS5A RASs emerged in Gt1a- infected patients between pre- and post-treatment. For the 3rd Gt1l-infected patient, NGS data was only available at pre-treatment. Typically, RAS in NS5A are located at positions 24, 28, 30, 31 and 93 as well as residue 58 (for Gt1a) [1]. However, these positions are polymorphic among HCV genotypes/subtypes. The Gt1l sequences in patients P1 and P3 between residues 28-31 were identical (MPRM; Table 1). Thus, at pre-treatment, both patients contained two documented RAS (Q30R+L31M). The   3rd gt1l-infected patient, P2, had one RAS (L31M). In a recent report, introducing Q30R+L31M into a Gt1a replicon confers high in vitro resistance for LDV and DCV (>23,400 and >4,452 increased EC50 values, respectively); for Velpatasvir, there is a 198-fold rise in EC50 for these substitutions [5]. In the context of Gt1a, L31M also increases EC50 values for these DAAs but to a lesser extent compared to Q30R+L31M. In the EAP cohort, there were also one non-responder and 3 responder-relapsers in the Gt4- infected group. From NGS analysis, the non-responder (P4) and one of the three responder- relapsers were infected with Gt4r (P5; Table 1); both patients originated from Somalia. The remaining responder-relapsers were Gt4d-infected. Similar to Gt1l-infected patients P1 and P3, no amino acid alterations were detected in NS5A between pre- and post-treatment for the Gt4r non-responder. The sequence between NS5A residues 28-32 in this patient was identical to that for patients P1 and P3 and thus contained a Q30R+L31M RAS doublet. However, a T282 RAS in NS5B, which is associated with SOF resistance [6], emerged in 100% of sequences at 1 week post-therapy (Figure 1). T282 remained dominant at week 5 post- treatment but at 49 weeks, T282 was not detected and all sequences encoded S282. A similar pattern of emergence and loss of T282 after failed SOF treatment has been reported in a Gt2b- infected patient [7]. The GT4r responder-relapser (P5) had a single NS5A RAS at pre- treatment (Q30R) and a further Y93H RAS emerged upon relapse. No amino acid changes were observed in NS5B. From the limited Gt1l sequences available (n=3), both double (Q30R+L31M) and single RAS (L31M) are present in published strains [8]. Combined with our data, NS5A position 30 is apparently polymorphic in Gt1l and thereby generates strains with double and single RAS, which could increase NS5A DAA resistance. As with Gt1l, there is a paucity of Gt4r sequence data. However, in a recently reported Gt4 cohort (n=44), Gt4r was detected in three subjects, two of whom did not achieve SVR [9]. The non-SVR Gt4r subjects had baseline RAS (M28M/V+Q30R+L31M), which remained the dominant sequences post-treatment. Moreover, a S282T mutation in NS5B emerged at post-treatment for one Gt4r patient. These data complement our findings and, given that the SOF-resistant S282T variant has emerged in 2/4   Gt4r-infected patients who did not achieve SVR, this substitution may arise with higher frequency in rare subtypes such as Gt4r. We conclude that certain Gt1l and Gt4r strains contain naturally occurring polymorphisms, which could contribute to resistance to NS5A inhibitors in patients with advanced liver disease. Since decompensated cirrhosis can lead to reduced response to DAAs [10], it is unclear whether extended duration of initial therapy might be advantageous in such patients nor whether patients with less liver damage would respond differently. Nevertheless, with the growing availability of DAAs, it is important that recommendations and guidance reflect the global situation to effectively control HCV infection. Since many DAA combinations for pan- genotypic HCV therapy include NS5A inhibitors, robust evidence is critical to evaluate their effectiveness against the global diversity of HCV subtypes and further trials may be needed in such cohorts to evaluate alternative regimens, perhaps including emerging protease inhibitor combinations. The absence of data in exotic HCV strains and the presence of variants associated with reduced treatment response pose a potential barrier to HCV eradication. Further studies investigating DAA efficacy in these and other strains found in LMICs in cirrhotic and non-cirrhotic patients are required. REFERENCES  [1] Zeuzem, S., Mizokami, M., Pianko, S., Mangia, A., Han, K.H., Martin, R. et al. NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome. J Hepatol. 2017; 66: 910-918. [2]   EASL recommendations on treatment of Hepatitis C 2016. J Hepatol. 2017; 66: 153-  194.  [3] Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S., McLauchlan, J., Mutimer, D.J. et al. Outcomes after successful direct-acting antiviral therapy for patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 65: 741-747. [4] Foster, G.R., Irving, W.L., Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S. et al. Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 64: 1224-1231.   [5] Wyles, D., Dvory-Sobol, H., Svarovskaia, E.S., Doehle, B.P., Martin, R., Afdhal, N.H. et al. Post-treatment resistance analysis of hepatitis C virus from phase II and III clinical trials of ledipasvir/sofosbuvir. J Hepatol. 2017; 66: 703-710. [6] Lam, A.M., Espiritu, C., Bansal, S., Micolochick Steuer, H.M., Niu, C., Zennou, V. et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother. 2012; 56: 3359-3368. [7] Hedskog, C., Dvory-Sobol, H., Gontcharova, V., Martin, R., Ouyang, W., Han, B. et al. Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy. J Viral Hepat. 2015; 22: 871-881. [8] Li, C., Njouom, R., Pepin, J., Nakano, T., Bennett, P., Pybus, O.G. et al. Characterization of full-length hepatitis C virus sequences for subtypes 1e, 1h and 1l, and a novel variant revealed Cameroon as an area in origin for genotype 1. J Gen Virol. 2013; 94: 1780-1790. [9] Abergel, A., Metivier, S., Samuel, D., Jiang, D., Kersey, K., Pang, P.S. et al. Ledipasvir plus sofosbuvir for 12 weeks in patients with hepatitis C genotype 4 infection. Hepatology. 2016; 64: 1049-1056. [10] Curry, M.P., O'Leary, J.G., Bzowej, N., Muir, A.J., Korenblat, K.M., Fenkel, J.M. et al. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. New Engl J Med. 2015; 373: 2618-2628.   Table 1. Outcomes of DAA treatment/retreatment and sequences at sites of NS5A RAS before and after treatment.   Position of NS5A RAS  HCV Gt Subjects/ Reference DAA Rx Rx Outcome (1) Rx Outcome (2) Timepoint 28 29 30 31 32 58 93 Gt1a Gt1a Ref     M P Q L P H Y Gt1b Gt1b Ref     L P R L P P Y     Gt1l  P1 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P2  SOF/DCV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M P Q M NA P P Y  P3 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y Gt4a Gt4a ref     V P L M P P Y    Gt4r  P4  SOF/LDV  Non-Resp Not re-treated Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P5 SOF/LDV/ RBV  Resp-Rel Not re-treated Pre-Rx Post-Rx M M P P R R L L P P P P Y H NA – sequence not available; Rx outcome (1) and (2) – initial (1) and retreatment (2) outcomes.   FIGURE LEGEND  Figure 1. Emergence and loss of RAS S282T in NS5B after therapy in patient P4. Boxes show the percentage reads of S282 and T282 from NGS data at pre- and post-treatment.   

Response to DAA Therapy in the NHS England Early Access Programme for Rare HCV Subtypes from Low and Middle Income Countries

Irving, William L.

Foster, Graham R.

Enlighten: Publications

Accepted Manuscript
Response to DAA Therapy in the NHS England Early Access Programme for
Rare HCV Subtypes from Low and Middle Income Countries
Ana da Silva Filipe, Vattipally Sreenu, Joseph Hughes, Elihu Aranday-Cortes,
William L Irving, Graham R. Foster, Kosh Agarwal, William Rosenberg,
Douglas Macdonald, Paul Richardson, Mark A. Aldersley, Martin Wiselka,
Andrew Ustianowski, John McLauchlan, Emma C. Thomson
PII: S0168-8278(17)32195-5
DOI: http://dx.doi.org/10.1016/j.jhep.2017.06.035
Reference: JHEPAT 6615
To appear in: Journal of Hepatology
Received Date: 28 June 2017
Accepted Date: 29 June 2017
Please cite this article as: da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W.L., Foster, G.R.,
Agarwal, K., Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M.A., Wiselka, M., Ustianowski, A.,
McLauchlan, J., Thomson, E.C., Response to DAA Therapy in the NHS England Early Access Programme for Rare
HCV Subtypes from Low and Middle Income Countries, Journal of Hepatology (2017), doi: http://dx.doi.org/
10.1016/j.jhep.2017.06.035
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
  
Response to DAA Therapy in the NHS England Early Access Programme for Rare 
HCV Subtypes from Low and Middle Income Countries 
 
 
Ana da Silva Filipe1, Vattipally Sreenu1, Joseph Hughes1, Elihu Aranday-Cortes1, William L 
Irving2, Graham R. Foster3, Kosh Agarwal4, William Rosenberg5, Douglas Macdonald5, Paul 
Richardson6, Mark A. Aldersley7, Martin Wiselka8, Andrew Ustianowski9, John McLauchlan1, 
Emma C. Thomson1 
1. MRC-University of Glasgow Centre for Virus Research, Glasgow, UK. 
 
2. Gastrointestinal and Liver Disorders Theme, NIHR Nottingham Biomedical Research Centre at the 
Nottingham University Hospitals NHS Trust and the University of Nottingham, UK. 
3. Blizard Institute, Queen Mary University of London, London, UK. 
 
4. Institute of Liver Studies, King's College Hospital Foundation Trust, London UK. 
 
5. Institute of Liver and Digestive Health, University College of London, London, United 
Kingdom. 
6. Royal Liverpool University NHS Trust, Liverpool, UK. 
 
7. Liver Unit, St James’s University Hospital, Leeds, UK. 
 
8. Infectious Diseases Unit, University Hospitals Leicester NHS Trust, Leicester, UK. 
 
9. Infectious Diseases, North Manchester General Hospital, Manchester, UK. 
 
Financial Support: This work was supported by grants from the Medical Research Foundation 
to HCV Research UK (C0365) and the UK Medical Research Council (MRC) to the STOP-HCV 
consortium (MR/K01532X/1). AS, VS, JH, EAC, JM and ECT were also supported by the MRC 
(MC.UU.12014/1) and ECT was funded by the Wellcome Trust (102789/Z/13/Z). We are also 
grateful to Gilead Sciences and Bristol Myers Squibb for financial support. 
Correspondence: Prof J McLauchlan, MRC-University of Glasgow Centre for Virus Research, 
Sir Michael Stoker Building, Garscube Campus, Glasgow G61 1QH, UK; E-mail,  
john.mclauchlan@glasgow.ac.uk; Tel no. +44 141 330 4028. 
Conflict of Interest: The authors disclose the following conflicts: WLI has received speaker 
and consultancy fees from Roche Products, Janssen Cilag and Novartis, educational grants 
  
from Boehringer Ingelheim, MSD and Gilead Sciences, and research grant support from 
GlaxoSmithKline, Pfizer, Gilead Sciences and Janssen Cilag; GRF has received research grants 
from Roche, Gilead Sciences, Springbank, and received speaker and consultancy fees from 
AbbVie, Gilead Sciences, MSD, GSK and Janssen Pharmaceuticals; KA has received speaker 
and consultancy fees from AbbVie, Achillion, Astellas, BMS, Gilead Sciences, GSK, Janssen, 
MSD, Novartis; WR is among the inventors and patent holders for the ELF® Test and receives 
consultancy fees from Siemens; MAA has been an advisory board member and in receipt of 
educational grants from Gilead Sciences; AU has received speaker fees and advisory board 
fees from Gilead Sciences, ViiV, Abbvie, MSD, Janssen and BMS; JM has received speaker and 
consultancy fees from Abbvie, Gilead Sciences and BMS. The remaining authors have no 
conflicts to declare. 
Author Contributions: The study was designed and led by AS, JM and ECT. EAC, VS and JH 
formed NGS and host genetic analysis. WLI collated and provided the clinical data from HCV 
Research UK. GRF, KA, WR, DM, PR, MAA, MW and AU consented the patients, provided clinical 
data and provided samples. All authors contributed to and commented on the drafting of the 
final manuscript. 
  
To the Editor: 
 
We read with interest “NS5A resistance-associated substitutions in patients with genotype 1 
hepatitis C virus: Prevalence and effect on treatment outcome” by Zeuzem et al. in the Journal 
of Hepatology, which indicated lower SVR rates for Gt1a and Gt1b strains carrying multiple 
resistance-associated substitutions (RASs) in NS5A [1]. Their study is very comprehensive 
but, as the authors note, it is largely restricted to HCV subtypes Gt1a and Gt1b, and does not 
represent subtypes from large geographical regions, including Africa. Indeed, current 
guidelines for treatment with direct-acting antivirals (DAAs) [2] are based on trial data for a 
limited selection of HCV genotypes, typically found in high-income countries. However, HCV is 
highly variable with natural polymorphisms at sites of DAA resistance. Our analysis of patients 
originating from low- and middle-income countries (LMICs), recruited into the NHS England 
Early Access Programme (EAP), has identified HCV subtypes, which contain naturally-occurring 
single and double RAS in NS5A that are associated with apparent poor response to approved 
DAAs. 
Previously, we described clinical outcomes from the EAP cohort, which provided IFN-free, DAA 
therapy to >800 HCV-infected patients with decompensated liver disease [3, 4]; all patients 
received sofosbuvir±ribavirin (SOF±RBV) combined with either ledipasvir (LDV) or daclatasvir 
(DCV). Among the Gt1-infected group, 28 patients did not achieve a SVR. From next 
generation sequence (NGS) analysis, 3 of these patients, designated as Gt1- or Gt1a-infected 
by the clinical sites, were infected with Gt1l. All 3 patients originated from Nigeria. The patients 
were retreated with DAAs for a further 24 weeks but again relapsed after therapy. In two Gt1l- 
infected patients (P1 and P3) where pre- and post-treatment samples were available, there 
were no sequence changes in either NS5A or NS5B. By contrast, NS5A RASs emerged in Gt1a- 
infected patients between pre- and post-treatment. For the 3rd Gt1l-infected patient, NGS data 
was only available at pre-treatment. 
Typically, RAS in NS5A are located at positions 24, 28, 30, 31 and 93 as well as residue 58 
(for Gt1a) [1]. However, these positions are polymorphic among HCV genotypes/subtypes. 
The Gt1l sequences in patients P1 and P3 between residues 28-31 were identical (MPRM; Table 
1). Thus, at pre-treatment, both patients contained two documented RAS (Q30R+L31M). The 
  
3rd gt1l-infected patient, P2, had one RAS (L31M). In a recent report, introducing Q30R+L31M 
into a Gt1a replicon confers high in vitro resistance for LDV and DCV (>23,400 and >4,452 
increased EC50 values, respectively); for Velpatasvir, there is a 198-fold rise in EC50 for these 
substitutions [5]. In the context of Gt1a, L31M also increases EC50 values for these DAAs but 
to a lesser extent compared to Q30R+L31M. 
In the EAP cohort, there were also one non-responder and 3 responder-relapsers in the Gt4- 
infected group. From NGS analysis, the non-responder (P4) and one of the three responder- 
relapsers were infected with Gt4r (P5; Table 1); both patients originated from Somalia. The 
remaining responder-relapsers were Gt4d-infected. Similar to Gt1l-infected patients P1 and 
P3, no amino acid alterations were detected in NS5A between pre- and post-treatment for the 
Gt4r non-responder. The sequence between NS5A residues 28-32 in this patient was identical 
to that for patients P1 and P3 and thus contained a Q30R+L31M RAS doublet. However, a 
T282 RAS in NS5B, which is associated with SOF resistance [6], emerged in 100% of 
sequences at 1 week post-therapy (Figure 1). T282 remained dominant at week 5 post- 
treatment but at 49 weeks, T282 was not detected and all sequences encoded S282. A similar 
pattern of emergence and loss of T282 after failed SOF treatment has been reported in a Gt2b- 
infected patient [7]. The GT4r responder-relapser (P5) had a single NS5A RAS at pre- 
treatment (Q30R) and a further Y93H RAS emerged upon relapse. No amino acid changes 
were observed in NS5B. 
From the limited Gt1l sequences available (n=3), both double (Q30R+L31M) and single RAS 
(L31M) are present in published strains [8]. Combined with our data, NS5A position 30 is 
apparently polymorphic in Gt1l and thereby generates strains with double and single RAS, 
which could increase NS5A DAA resistance. As with Gt1l, there is a paucity of Gt4r sequence 
data. However, in a recently reported Gt4 cohort (n=44), Gt4r was detected in three subjects, 
two of whom did not achieve SVR [9]. The non-SVR Gt4r subjects had baseline RAS 
(M28M/V+Q30R+L31M), which remained the dominant sequences post-treatment. Moreover, 
a S282T mutation in NS5B emerged at post-treatment for one Gt4r patient. These data 
complement our findings and, given that the SOF-resistant S282T variant has emerged in 2/4 
  
Gt4r-infected patients who did not achieve SVR, this substitution may arise with higher 
frequency in rare subtypes such as Gt4r. 
We conclude that certain Gt1l and Gt4r strains contain naturally occurring polymorphisms, 
which could contribute to resistance to NS5A inhibitors in patients with advanced liver disease. 
Since decompensated cirrhosis can lead to reduced response to DAAs [10], it is unclear 
whether extended duration of initial therapy might be advantageous in such patients nor 
whether patients with less liver damage would respond differently. Nevertheless, with the 
growing availability of DAAs, it is important that recommendations and guidance reflect the 
global situation to effectively control HCV infection. Since many DAA combinations for pan- 
genotypic HCV therapy include NS5A inhibitors, robust evidence is critical to evaluate their 
effectiveness against the global diversity of HCV subtypes and further trials may be needed in 
such cohorts to evaluate alternative regimens, perhaps including emerging protease inhibitor 
combinations. The absence of data in exotic HCV strains and the presence of variants 
associated with reduced treatment response pose a potential barrier to HCV eradication. 
Further studies investigating DAA efficacy in these and other strains found in LMICs in cirrhotic 
and non-cirrhotic patients are required. 
REFERENCES 
 
[1] Zeuzem, S., Mizokami, M., Pianko, S., Mangia, A., Han, K.H., Martin, R. et al. NS5A 
resistance-associated substitutions in patients with genotype 1 hepatitis C 
virus: Prevalence and effect on treatment outcome. J Hepatol. 2017; 66: 910-918. 
[2]   EASL recommendations on treatment of Hepatitis C 2016. J Hepatol. 2017; 66: 153- 
 
194. 
 
[3] Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S., McLauchlan, J., Mutimer, D.J. et 
al. Outcomes after successful direct-acting antiviral therapy for patients with 
chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 65: 741-747. 
[4] Foster, G.R., Irving, W.L., Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S. et al. 
Impact of direct acting antiviral therapy in patients with chronic hepatitis C and 
decompensated cirrhosis. J Hepatol. 2016; 64: 1224-1231. 
  
[5] Wyles, D., Dvory-Sobol, H., Svarovskaia, E.S., Doehle, B.P., Martin, R., Afdhal, N.H. et 
al. Post-treatment resistance analysis of hepatitis C virus from phase II and III 
clinical trials of ledipasvir/sofosbuvir. J Hepatol. 2017; 66: 703-710. 
[6] Lam, A.M., Espiritu, C., Bansal, S., Micolochick Steuer, H.M., Niu, C., Zennou, V. et al. 
Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of 
hepatitis C virus. Antimicrob Agents Chemother. 2012; 56: 3359-3368. 
[7] Hedskog, C., Dvory-Sobol, H., Gontcharova, V., Martin, R., Ouyang, W., Han, B. et al. 
Evolution of the HCV viral population from a patient with S282T detected at 
relapse after sofosbuvir monotherapy. J Viral Hepat. 2015; 22: 871-881. 
[8] Li, C., Njouom, R., Pepin, J., Nakano, T., Bennett, P., Pybus, O.G. et al. 
Characterization of full-length hepatitis C virus sequences for subtypes 1e, 1h 
and 1l, and a novel variant revealed Cameroon as an area in origin for genotype 
1. J Gen Virol. 2013; 94: 1780-1790. 
[9] Abergel, A., Metivier, S., Samuel, D., Jiang, D., Kersey, K., Pang, P.S. et al. Ledipasvir 
plus sofosbuvir for 12 weeks in patients with hepatitis C genotype 4 infection. 
Hepatology. 2016; 64: 1049-1056. 
[10] Curry, M.P., O'Leary, J.G., Bzowej, N., Muir, A.J., Korenblat, K.M., Fenkel, J.M. et al. 
Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. 
New Engl J Med. 2015; 373: 2618-2628. 
  
Table 1. Outcomes of DAA treatment/retreatment and sequences at sites of NS5A 
RAS before and after treatment. 
 
 Position of NS5A RAS  
HCV Gt 
Subjects/ 
Reference 
DAA Rx 
Rx Outcome 
(1) 
Rx Outcome 
(2) 
Timepoint 28 29 30 31 32 58 93 
Gt1a Gt1a Ref     M P Q L P H Y 
Gt1b Gt1b Ref     L P R L P P Y 
 
 
 
 
Gt1l 
 
P1 
SOF/LDV/ 
RBV 
 
Resp-Rel 
 
Resp-Rel 
Pre-Rx 
Post-Rx 
M 
M 
P 
P 
R 
R 
M 
M 
P 
P 
P 
P 
Y 
Y 
 
P2 
 
SOF/DCV 
 
Resp-Rel 
 
Resp-Rel 
Pre-Rx 
Post-Rx 
M P Q M 
NA 
P P Y 
 
P3 
SOF/LDV/ 
RBV 
 
Resp-Rel 
 
Resp-Rel 
Pre-Rx 
Post-Rx 
M 
M 
P 
P 
R 
R 
M 
M 
P 
P 
P 
P 
Y 
Y 
Gt4a Gt4a ref     V P L M P P Y 
 
 
 
Gt4r 
 
P4 
 
SOF/LDV 
 
Non-Resp 
Not 
re-treated 
Pre-Rx 
Post-Rx 
M 
M 
P 
P 
R 
R 
M 
M 
P 
P 
P 
P 
Y 
Y 
 
P5 
SOF/LDV/ 
RBV 
 
Resp-Rel 
Not 
re-treated 
Pre-Rx 
Post-Rx 
M 
M 
P 
P 
R 
R 
L 
L 
P 
P 
P 
P 
Y 
H 
NA – sequence not available; Rx outcome (1) and (2) – initial (1) and retreatment (2) 
outcomes. 
  
FIGURE LEGEND 
 
Figure 1. Emergence and loss of RAS S282T in NS5B after therapy in patient P4. Boxes show 
the percentage reads of S282 and T282 from NGS data at pre- and post-treatment. 
 
 


Response to DAA therapy in the NHS England early access programme for rare HCV subtypes from low and middle income countries

Enlighten

Accepted ManuscriptResponse to DAA Therapy in the NHS England Early Access Programme forRare HCV Subtypes from Low and Middle Income CountriesAna da Silva Filipe, Vattipally Sreenu, Joseph Hughes, Elihu Aranday-Cortes,William L Irving, Graham R. Foster, Kosh Agarwal, William Rosenberg,Douglas Macdonald, Paul Richardson, Mark A. Aldersley, Martin Wiselka,Andrew Ustianowski, John McLauchlan, Emma C. ThomsonPII: S0168-8278(17)32195-5DOI: http://dx.doi.org/10.1016/j.jhep.2017.06.035Reference: JHEPAT 6615To appear in: Journal of HepatologyReceived Date: 28 June 2017Accepted Date: 29 June 2017Please cite this article as: da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W.L., Foster, G.R.,Agarwal, K., Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M.A., Wiselka, M., Ustianowski, A.,McLauchlan, J., Thomson, E.C., Response to DAA Therapy in the NHS England Early Access Programme for RareHCV Subtypes from Low and Middle Income Countries, Journal of Hepatology (2017), doi: http://dx.doi.org/10.1016/j.jhep.2017.06.035This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  Response to DAA Therapy in the NHS England Early Access Programme for Rare HCV Subtypes from Low and Middle Income Countries   Ana da Silva Filipe1, Vattipally Sreenu1, Joseph Hughes1, Elihu Aranday-Cortes1, William L Irving2, Graham R. Foster3, Kosh Agarwal4, William Rosenberg5, Douglas Macdonald5, Paul Richardson6, Mark A. Aldersley7, Martin Wiselka8, Andrew Ustianowski9, John McLauchlan1, Emma C. Thomson1 1. MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.  2. Gastrointestinal and Liver Disorders Theme, NIHR Nottingham Biomedical Research Centre at the Nottingham University Hospitals NHS Trust and the University of Nottingham, UK. 3. Blizard Institute, Queen Mary University of London, London, UK.  4. Institute of Liver Studies, King's College Hospital Foundation Trust, London UK.  5. Institute of Liver and Digestive Health, University College of London, London, United Kingdom. 6. Royal Liverpool University NHS Trust, Liverpool, UK.  7. Liver Unit, St James’s University Hospital, Leeds, UK.  8. Infectious Diseases Unit, University Hospitals Leicester NHS Trust, Leicester, UK.  9. Infectious Diseases, North Manchester General Hospital, Manchester, UK.  Financial Support: This work was supported by grants from the Medical Research Foundation to HCV Research UK (C0365) and the UK Medical Research Council (MRC) to the STOP-HCV consortium (MR/K01532X/1). AS, VS, JH, EAC, JM and ECT were also supported by the MRC (MC.UU.12014/1) and ECT was funded by the Wellcome Trust (102789/Z/13/Z). We are also grateful to Gilead Sciences and Bristol Myers Squibb for financial support. Correspondence: Prof J McLauchlan, MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, Glasgow G61 1QH, UK; E-mail,  john.mclauchlan@glasgow.ac.uk; Tel no. +44 141 330 4028. Conflict of Interest: The authors disclose the following conflicts: WLI has received speaker and consultancy fees from Roche Products, Janssen Cilag and Novartis, educational grants   from Boehringer Ingelheim, MSD and Gilead Sciences, and research grant support from GlaxoSmithKline, Pfizer, Gilead Sciences and Janssen Cilag; GRF has received research grants from Roche, Gilead Sciences, Springbank, and received speaker and consultancy fees from AbbVie, Gilead Sciences, MSD, GSK and Janssen Pharmaceuticals; KA has received speaker and consultancy fees from AbbVie, Achillion, Astellas, BMS, Gilead Sciences, GSK, Janssen, MSD, Novartis; WR is among the inventors and patent holders for the ELF® Test and receives consultancy fees from Siemens; MAA has been an advisory board member and in receipt of educational grants from Gilead Sciences; AU has received speaker fees and advisory board fees from Gilead Sciences, ViiV, Abbvie, MSD, Janssen and BMS; JM has received speaker and consultancy fees from Abbvie, Gilead Sciences and BMS. The remaining authors have no conflicts to declare. Author Contributions: The study was designed and led by AS, JM and ECT. EAC, VS and JH formed NGS and host genetic analysis. WLI collated and provided the clinical data from HCV Research UK. GRF, KA, WR, DM, PR, MAA, MW and AU consented the patients, provided clinical data and provided samples. All authors contributed to and commented on the drafting of the final manuscript.   To the Editor:  We read with interest “NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome” by Zeuzem et al. in the Journal of Hepatology, which indicated lower SVR rates for Gt1a and Gt1b strains carrying multiple resistance-associated substitutions (RASs) in NS5A [1]. Their study is very comprehensive but, as the authors note, it is largely restricted to HCV subtypes Gt1a and Gt1b, and does not represent subtypes from large geographical regions, including Africa. Indeed, current guidelines for treatment with direct-acting antivirals (DAAs) [2] are based on trial data for a limited selection of HCV genotypes, typically found in high-income countries. However, HCV is highly variable with natural polymorphisms at sites of DAA resistance. Our analysis of patients originating from low- and middle-income countries (LMICs), recruited into the NHS England Early Access Programme (EAP), has identified HCV subtypes, which contain naturally-occurring single and double RAS in NS5A that are associated with apparent poor response to approved DAAs. Previously, we described clinical outcomes from the EAP cohort, which provided IFN-free, DAA therapy to >800 HCV-infected patients with decompensated liver disease [3, 4]; all patients received sofosbuvir±ribavirin (SOF±RBV) combined with either ledipasvir (LDV) or daclatasvir (DCV). Among the Gt1-infected group, 28 patients did not achieve a SVR. From next generation sequence (NGS) analysis, 3 of these patients, designated as Gt1- or Gt1a-infected by the clinical sites, were infected with Gt1l. All 3 patients originated from Nigeria. The patients were retreated with DAAs for a further 24 weeks but again relapsed after therapy. In two Gt1l- infected patients (P1 and P3) where pre- and post-treatment samples were available, there were no sequence changes in either NS5A or NS5B. By contrast, NS5A RASs emerged in Gt1a- infected patients between pre- and post-treatment. For the 3rd Gt1l-infected patient, NGS data was only available at pre-treatment. Typically, RAS in NS5A are located at positions 24, 28, 30, 31 and 93 as well as residue 58 (for Gt1a) [1]. However, these positions are polymorphic among HCV genotypes/subtypes. The Gt1l sequences in patients P1 and P3 between residues 28-31 were identical (MPRM; Table 1). Thus, at pre-treatment, both patients contained two documented RAS (Q30R+L31M). The   3rd gt1l-infected patient, P2, had one RAS (L31M). In a recent report, introducing Q30R+L31M into a Gt1a replicon confers high in vitro resistance for LDV and DCV (>23,400 and >4,452 increased EC50 values, respectively); for Velpatasvir, there is a 198-fold rise in EC50 for these substitutions [5]. In the context of Gt1a, L31M also increases EC50 values for these DAAs but to a lesser extent compared to Q30R+L31M. In the EAP cohort, there were also one non-responder and 3 responder-relapsers in the Gt4- infected group. From NGS analysis, the non-responder (P4) and one of the three responder- relapsers were infected with Gt4r (P5; Table 1); both patients originated from Somalia. The remaining responder-relapsers were Gt4d-infected. Similar to Gt1l-infected patients P1 and P3, no amino acid alterations were detected in NS5A between pre- and post-treatment for the Gt4r non-responder. The sequence between NS5A residues 28-32 in this patient was identical to that for patients P1 and P3 and thus contained a Q30R+L31M RAS doublet. However, a T282 RAS in NS5B, which is associated with SOF resistance [6], emerged in 100% of sequences at 1 week post-therapy (Figure 1). T282 remained dominant at week 5 post- treatment but at 49 weeks, T282 was not detected and all sequences encoded S282. A similar pattern of emergence and loss of T282 after failed SOF treatment has been reported in a Gt2b- infected patient [7]. The GT4r responder-relapser (P5) had a single NS5A RAS at pre- treatment (Q30R) and a further Y93H RAS emerged upon relapse. No amino acid changes were observed in NS5B. From the limited Gt1l sequences available (n=3), both double (Q30R+L31M) and single RAS (L31M) are present in published strains [8]. Combined with our data, NS5A position 30 is apparently polymorphic in Gt1l and thereby generates strains with double and single RAS, which could increase NS5A DAA resistance. As with Gt1l, there is a paucity of Gt4r sequence data. However, in a recently reported Gt4 cohort (n=44), Gt4r was detected in three subjects, two of whom did not achieve SVR [9]. The non-SVR Gt4r subjects had baseline RAS (M28M/V+Q30R+L31M), which remained the dominant sequences post-treatment. Moreover, a S282T mutation in NS5B emerged at post-treatment for one Gt4r patient. These data complement our findings and, given that the SOF-resistant S282T variant has emerged in 2/4   Gt4r-infected patients who did not achieve SVR, this substitution may arise with higher frequency in rare subtypes such as Gt4r. We conclude that certain Gt1l and Gt4r strains contain naturally occurring polymorphisms, which could contribute to resistance to NS5A inhibitors in patients with advanced liver disease. Since decompensated cirrhosis can lead to reduced response to DAAs [10], it is unclear whether extended duration of initial therapy might be advantageous in such patients nor whether patients with less liver damage would respond differently. Nevertheless, with the growing availability of DAAs, it is important that recommendations and guidance reflect the global situation to effectively control HCV infection. Since many DAA combinations for pan- genotypic HCV therapy include NS5A inhibitors, robust evidence is critical to evaluate their effectiveness against the global diversity of HCV subtypes and further trials may be needed in such cohorts to evaluate alternative regimens, perhaps including emerging protease inhibitor combinations. The absence of data in exotic HCV strains and the presence of variants associated with reduced treatment response pose a potential barrier to HCV eradication. Further studies investigating DAA efficacy in these and other strains found in LMICs in cirrhotic and non-cirrhotic patients are required. REFERENCES  [1] Zeuzem, S., Mizokami, M., Pianko, S., Mangia, A., Han, K.H., Martin, R. et al. NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome. J Hepatol. 2017; 66: 910-918. [2]   EASL recommendations on treatment of Hepatitis C 2016. J Hepatol. 2017; 66: 153-  194.  [3] Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S., McLauchlan, J., Mutimer, D.J. et al. Outcomes after successful direct-acting antiviral therapy for patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 65: 741-747. [4] Foster, G.R., Irving, W.L., Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S. et al. Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 64: 1224-1231.   [5] Wyles, D., Dvory-Sobol, H., Svarovskaia, E.S., Doehle, B.P., Martin, R., Afdhal, N.H. et al. Post-treatment resistance analysis of hepatitis C virus from phase II and III clinical trials of ledipasvir/sofosbuvir. J Hepatol. 2017; 66: 703-710. [6] Lam, A.M., Espiritu, C., Bansal, S., Micolochick Steuer, H.M., Niu, C., Zennou, V. et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother. 2012; 56: 3359-3368. [7] Hedskog, C., Dvory-Sobol, H., Gontcharova, V., Martin, R., Ouyang, W., Han, B. et al. Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy. J Viral Hepat. 2015; 22: 871-881. [8] Li, C., Njouom, R., Pepin, J., Nakano, T., Bennett, P., Pybus, O.G. et al. Characterization of full-length hepatitis C virus sequences for subtypes 1e, 1h and 1l, and a novel variant revealed Cameroon as an area in origin for genotype 1. J Gen Virol. 2013; 94: 1780-1790. [9] Abergel, A., Metivier, S., Samuel, D., Jiang, D., Kersey, K., Pang, P.S. et al. Ledipasvir plus sofosbuvir for 12 weeks in patients with hepatitis C genotype 4 infection. Hepatology. 2016; 64: 1049-1056. [10] Curry, M.P., O'Leary, J.G., Bzowej, N., Muir, A.J., Korenblat, K.M., Fenkel, J.M. et al. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. New Engl J Med. 2015; 373: 2618-2628.   Table 1. Outcomes of DAA treatment/retreatment and sequences at sites of NS5A RAS before and after treatment.   Position of NS5A RAS  HCV Gt Subjects/ Reference DAA Rx Rx Outcome (1) Rx Outcome (2) Timepoint 28 29 30 31 32 58 93 Gt1a Gt1a Ref     M P Q L P H Y Gt1b Gt1b Ref     L P R L P P Y     Gt1l  P1 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P2  SOF/DCV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M P Q M NA P P Y  P3 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y Gt4a Gt4a ref     V P L M P P Y    Gt4r  P4  SOF/LDV  Non-Resp Not re-treated Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P5 SOF/LDV/ RBV  Resp-Rel Not re-treated Pre-Rx Post-Rx M M P P R R L L P P P P Y H NA – sequence not available; Rx outcome (1) and (2) – initial (1) and retreatment (2) outcomes.   FIGURE LEGEND  Figure 1. Emergence and loss of RAS S282T in NS5B after therapy in patient P4. Boxes show the percentage reads of S282 and T282 from NGS data at pre- and post-treatment.   

                       King’s Research Portal  DOI:10.1016/j.jhep.2017.06.035Document VersionPeer reviewed versionLink to publication record in King's Research PortalCitation for published version (APA):da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W. L., Foster, G. R., Agarwal, K.,Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M. A., Wiselka, M., Ustianowski, A., McLauchlan, J.,& Thomson, E. C. (2017). Response to DAA Therapy in the NHS England Early Access Programme for RareHCV Subtypes from Low and Middle Income Countries. Journal of Hepatology.https://doi.org/10.1016/j.jhep.2017.06.035Citing this paperPlease note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this maydiffer from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination,volume/issue, and date of publication details. 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Oct. 2023Accepted ManuscriptResponse to DAA Therapy in the NHS England Early Access Programme forRare HCV Subtypes from Low and Middle Income CountriesAna da Silva Filipe, Vattipally Sreenu, Joseph Hughes, Elihu Aranday-Cortes,William L Irving, Graham R. Foster, Kosh Agarwal, William Rosenberg,Douglas Macdonald, Paul Richardson, Mark A. Aldersley, Martin Wiselka,Andrew Ustianowski, John McLauchlan, Emma C. ThomsonPII: S0168-8278(17)32195-5DOI: http://dx.doi.org/10.1016/j.jhep.2017.06.035Reference: JHEPAT 6615To appear in: Journal of HepatologyReceived Date: 28 June 2017Accepted Date: 29 June 2017Please cite this article as: da Silva Filipe, A., Sreenu, V., Hughes, J., Aranday-Cortes, E., Irving, W.L., Foster, G.R.,Agarwal, K., Rosenberg, W., Macdonald, D., Richardson, P., Aldersley, M.A., Wiselka, M., Ustianowski, A.,McLauchlan, J., Thomson, E.C., Response to DAA Therapy in the NHS England Early Access Programme for RareHCV Subtypes from Low and Middle Income Countries, Journal of Hepatology (2017), doi: http://dx.doi.org/10.1016/j.jhep.2017.06.035This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  Response to DAA Therapy in the NHS England Early Access Programme for Rare HCV Subtypes from Low and Middle Income Countries   Ana da Silva Filipe1, Vattipally Sreenu1, Joseph Hughes1, Elihu Aranday-Cortes1, William L Irving2, Graham R. Foster3, Kosh Agarwal4, William Rosenberg5, Douglas Macdonald5, Paul Richardson6, Mark A. Aldersley7, Martin Wiselka8, Andrew Ustianowski9, John McLauchlan1, Emma C. Thomson1 1. MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.  2. Gastrointestinal and Liver Disorders Theme, NIHR Nottingham Biomedical Research Centre at the Nottingham University Hospitals NHS Trust and the University of Nottingham, UK. 3. Blizard Institute, Queen Mary University of London, London, UK.  4. Institute of Liver Studies, King's College Hospital Foundation Trust, London UK.  5. Institute of Liver and Digestive Health, University College of London, London, United Kingdom. 6. Royal Liverpool University NHS Trust, Liverpool, UK.  7. Liver Unit, St James’s University Hospital, Leeds, UK.  8. Infectious Diseases Unit, University Hospitals Leicester NHS Trust, Leicester, UK.  9. Infectious Diseases, North Manchester General Hospital, Manchester, UK.  Financial Support: This work was supported by grants from the Medical Research Foundation to HCV Research UK (C0365) and the UK Medical Research Council (MRC) to the STOP-HCV consortium (MR/K01532X/1). AS, VS, JH, EAC, JM and ECT were also supported by the MRC (MC.UU.12014/1) and ECT was funded by the Wellcome Trust (102789/Z/13/Z). We are also grateful to Gilead Sciences and Bristol Myers Squibb for financial support. Correspondence: Prof J McLauchlan, MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, Glasgow G61 1QH, UK; E-mail,  john.mclauchlan@glasgow.ac.uk; Tel no. +44 141 330 4028. Conflict of Interest: The authors disclose the following conflicts: WLI has received speaker and consultancy fees from Roche Products, Janssen Cilag and Novartis, educational grants   from Boehringer Ingelheim, MSD and Gilead Sciences, and research grant support from GlaxoSmithKline, Pfizer, Gilead Sciences and Janssen Cilag; GRF has received research grants from Roche, Gilead Sciences, Springbank, and received speaker and consultancy fees from AbbVie, Gilead Sciences, MSD, GSK and Janssen Pharmaceuticals; KA has received speaker and consultancy fees from AbbVie, Achillion, Astellas, BMS, Gilead Sciences, GSK, Janssen, MSD, Novartis; WR is among the inventors and patent holders for the ELF® Test and receives consultancy fees from Siemens; MAA has been an advisory board member and in receipt of educational grants from Gilead Sciences; AU has received speaker fees and advisory board fees from Gilead Sciences, ViiV, Abbvie, MSD, Janssen and BMS; JM has received speaker and consultancy fees from Abbvie, Gilead Sciences and BMS. The remaining authors have no conflicts to declare. Author Contributions: The study was designed and led by AS, JM and ECT. EAC, VS and JH formed NGS and host genetic analysis. WLI collated and provided the clinical data from HCV Research UK. GRF, KA, WR, DM, PR, MAA, MW and AU consented the patients, provided clinical data and provided samples. All authors contributed to and commented on the drafting of the final manuscript.   To the Editor:  We read with interest “NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome” by Zeuzem et al. in the Journal of Hepatology, which indicated lower SVR rates for Gt1a and Gt1b strains carrying multiple resistance-associated substitutions (RASs) in NS5A [1]. Their study is very comprehensive but, as the authors note, it is largely restricted to HCV subtypes Gt1a and Gt1b, and does not represent subtypes from large geographical regions, including Africa. Indeed, current guidelines for treatment with direct-acting antivirals (DAAs) [2] are based on trial data for a limited selection of HCV genotypes, typically found in high-income countries. However, HCV is highly variable with natural polymorphisms at sites of DAA resistance. Our analysis of patients originating from low- and middle-income countries (LMICs), recruited into the NHS England Early Access Programme (EAP), has identified HCV subtypes, which contain naturally-occurring single and double RAS in NS5A that are associated with apparent poor response to approved DAAs. Previously, we described clinical outcomes from the EAP cohort, which provided IFN-free, DAA therapy to >800 HCV-infected patients with decompensated liver disease [3, 4]; all patients received sofosbuvir±ribavirin (SOF±RBV) combined with either ledipasvir (LDV) or daclatasvir (DCV). Among the Gt1-infected group, 28 patients did not achieve a SVR. From next generation sequence (NGS) analysis, 3 of these patients, designated as Gt1- or Gt1a-infected by the clinical sites, were infected with Gt1l. All 3 patients originated from Nigeria. The patients were retreated with DAAs for a further 24 weeks but again relapsed after therapy. In two Gt1l- infected patients (P1 and P3) where pre- and post-treatment samples were available, there were no sequence changes in either NS5A or NS5B. By contrast, NS5A RASs emerged in Gt1a- infected patients between pre- and post-treatment. For the 3rd Gt1l-infected patient, NGS data was only available at pre-treatment. Typically, RAS in NS5A are located at positions 24, 28, 30, 31 and 93 as well as residue 58 (for Gt1a) [1]. However, these positions are polymorphic among HCV genotypes/subtypes. The Gt1l sequences in patients P1 and P3 between residues 28-31 were identical (MPRM; Table 1). Thus, at pre-treatment, both patients contained two documented RAS (Q30R+L31M). The   3rd gt1l-infected patient, P2, had one RAS (L31M). In a recent report, introducing Q30R+L31M into a Gt1a replicon confers high in vitro resistance for LDV and DCV (>23,400 and >4,452 increased EC50 values, respectively); for Velpatasvir, there is a 198-fold rise in EC50 for these substitutions [5]. In the context of Gt1a, L31M also increases EC50 values for these DAAs but to a lesser extent compared to Q30R+L31M. In the EAP cohort, there were also one non-responder and 3 responder-relapsers in the Gt4- infected group. From NGS analysis, the non-responder (P4) and one of the three responder- relapsers were infected with Gt4r (P5; Table 1); both patients originated from Somalia. The remaining responder-relapsers were Gt4d-infected. Similar to Gt1l-infected patients P1 and P3, no amino acid alterations were detected in NS5A between pre- and post-treatment for the Gt4r non-responder. The sequence between NS5A residues 28-32 in this patient was identical to that for patients P1 and P3 and thus contained a Q30R+L31M RAS doublet. However, a T282 RAS in NS5B, which is associated with SOF resistance [6], emerged in 100% of sequences at 1 week post-therapy (Figure 1). T282 remained dominant at week 5 post- treatment but at 49 weeks, T282 was not detected and all sequences encoded S282. A similar pattern of emergence and loss of T282 after failed SOF treatment has been reported in a Gt2b- infected patient [7]. The GT4r responder-relapser (P5) had a single NS5A RAS at pre- treatment (Q30R) and a further Y93H RAS emerged upon relapse. No amino acid changes were observed in NS5B. From the limited Gt1l sequences available (n=3), both double (Q30R+L31M) and single RAS (L31M) are present in published strains [8]. Combined with our data, NS5A position 30 is apparently polymorphic in Gt1l and thereby generates strains with double and single RAS, which could increase NS5A DAA resistance. As with Gt1l, there is a paucity of Gt4r sequence data. However, in a recently reported Gt4 cohort (n=44), Gt4r was detected in three subjects, two of whom did not achieve SVR [9]. The non-SVR Gt4r subjects had baseline RAS (M28M/V+Q30R+L31M), which remained the dominant sequences post-treatment. Moreover, a S282T mutation in NS5B emerged at post-treatment for one Gt4r patient. These data complement our findings and, given that the SOF-resistant S282T variant has emerged in 2/4   Gt4r-infected patients who did not achieve SVR, this substitution may arise with higher frequency in rare subtypes such as Gt4r. We conclude that certain Gt1l and Gt4r strains contain naturally occurring polymorphisms, which could contribute to resistance to NS5A inhibitors in patients with advanced liver disease. Since decompensated cirrhosis can lead to reduced response to DAAs [10], it is unclear whether extended duration of initial therapy might be advantageous in such patients nor whether patients with less liver damage would respond differently. Nevertheless, with the growing availability of DAAs, it is important that recommendations and guidance reflect the global situation to effectively control HCV infection. Since many DAA combinations for pan- genotypic HCV therapy include NS5A inhibitors, robust evidence is critical to evaluate their effectiveness against the global diversity of HCV subtypes and further trials may be needed in such cohorts to evaluate alternative regimens, perhaps including emerging protease inhibitor combinations. The absence of data in exotic HCV strains and the presence of variants associated with reduced treatment response pose a potential barrier to HCV eradication. Further studies investigating DAA efficacy in these and other strains found in LMICs in cirrhotic and non-cirrhotic patients are required. REFERENCES  [1] Zeuzem, S., Mizokami, M., Pianko, S., Mangia, A., Han, K.H., Martin, R. et al. NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome. J Hepatol. 2017; 66: 910-918. [2]   EASL recommendations on treatment of Hepatitis C 2016. J Hepatol. 2017; 66: 153-  194.  [3] Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S., McLauchlan, J., Mutimer, D.J. et al. Outcomes after successful direct-acting antiviral therapy for patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 65: 741-747. [4] Foster, G.R., Irving, W.L., Cheung, M.C., Walker, A.J., Hudson, B.E., Verma, S. et al. Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016; 64: 1224-1231.   [5] Wyles, D., Dvory-Sobol, H., Svarovskaia, E.S., Doehle, B.P., Martin, R., Afdhal, N.H. et al. Post-treatment resistance analysis of hepatitis C virus from phase II and III clinical trials of ledipasvir/sofosbuvir. J Hepatol. 2017; 66: 703-710. [6] Lam, A.M., Espiritu, C., Bansal, S., Micolochick Steuer, H.M., Niu, C., Zennou, V. et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother. 2012; 56: 3359-3368. [7] Hedskog, C., Dvory-Sobol, H., Gontcharova, V., Martin, R., Ouyang, W., Han, B. et al. Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy. J Viral Hepat. 2015; 22: 871-881. [8] Li, C., Njouom, R., Pepin, J., Nakano, T., Bennett, P., Pybus, O.G. et al. Characterization of full-length hepatitis C virus sequences for subtypes 1e, 1h and 1l, and a novel variant revealed Cameroon as an area in origin for genotype 1. J Gen Virol. 2013; 94: 1780-1790. [9] Abergel, A., Metivier, S., Samuel, D., Jiang, D., Kersey, K., Pang, P.S. et al. Ledipasvir plus sofosbuvir for 12 weeks in patients with hepatitis C genotype 4 infection. Hepatology. 2016; 64: 1049-1056. [10] Curry, M.P., O'Leary, J.G., Bzowej, N., Muir, A.J., Korenblat, K.M., Fenkel, J.M. et al. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. New Engl J Med. 2015; 373: 2618-2628.   Table 1. Outcomes of DAA treatment/retreatment and sequences at sites of NS5A RAS before and after treatment.   Position of NS5A RAS  HCV Gt Subjects/ Reference DAA Rx Rx Outcome (1) Rx Outcome (2) Timepoint 28 29 30 31 32 58 93 Gt1a Gt1a Ref     M P Q L P H Y Gt1b Gt1b Ref     L P R L P P Y     Gt1l  P1 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P2  SOF/DCV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M P Q M NA P P Y  P3 SOF/LDV/ RBV  Resp-Rel  Resp-Rel Pre-Rx Post-Rx M M P P R R M M P P P P Y Y Gt4a Gt4a ref     V P L M P P Y    Gt4r  P4  SOF/LDV  Non-Resp Not re-treated Pre-Rx Post-Rx M M P P R R M M P P P P Y Y  P5 SOF/LDV/ RBV  Resp-Rel Not re-treated Pre-Rx Post-Rx M M P P R R L L P P P P Y H NA – sequence not available; Rx outcome (1) and (2) – initial (1) and retreatment (2) outcomes.   FIGURE LEGEND  Figure 1. Emergence and loss of RAS S282T in NS5B after therapy in patient P4. Boxes show the percentage reads of S282 and T282 from NGS data at pre- and post-treatment.   

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Response to DAA Therapy in the NHS England Early Access Programme for Rare HCV Subtypes from Low and Middle Income Countries

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