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Journal of Gastroenterology

, Volume 54, Issue 5, pp 449–458 | Cite as

Combinations of two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents are effective for hepatitis C virus with NS5A-P32 deletion in humanized-liver mice

  • Akira Doi
  • Hayato Hikita
  • Yugo Kai
  • Yuki Tahata
  • Yoshinobu Saito
  • Tasuku Nakabori
  • Ryoko Yamada
  • Takahiro Kodama
  • Ryotaro Sakamori
  • Asako Murayama
  • Sayuri Nitta
  • Yasuhiro Asahina
  • Hiroshi Suemizu
  • Tomohide Tatsumi
  • Takanobu Kato
  • Tetsuo TakeharaEmail author
Original Article—Liver, Pancreas, and Biliary Tract

Abstract

Background

The emergence of a deletion mutant at hepatitis C virus (HCV) NS5A-P32 (P32del) has recently been reported in a subset of chronic hepatitis C patients who experience virologic failure after direct-acting antiviral drug (DAA) treatment. This mutation confers extremely high resistance to NS5A inhibitors. No effective treatment has been established for cases with this mutation.

Methods

We used a JFH1-based recombinant virus with NS5A from a genotype 1b strain to introduce a P32del mutation. We inoculated human hepatocyte chimeric mice with sera from a patient with ledipasvir/sofosbuvir therapy failure carrying a genotype 1b HCV with NS5A L31M and P32del or from a DAA-naïve patient carrying wild-type virus.

Results

JFH1-based chimeric viruses with P32del showed sufficient levels of replication for in vitro assay despite the suppression of viral growth and infectious virus production. Variants with P32del exhibited severe resistance to all tested NS5A inhibitors, including daclatasvir, ledipasvir, elbasvir and velpatasvir, but were as susceptible to NS3/4A inhibitors, NS5B inhibitors, interferon alfa-2b, and ribavirin as wild-type viruses in the in vitro assay. The P32del mutant virus caused persistent infection in all inoculated chimeric mice with high viral titer and frequency. The virus was resistant to the ledipasvir/GS-558093 (a nucleotide analog inhibitor of NS5B polymerase) regimen but susceptible to either simeprevir plus GS-558093 or peg-interferon alfa-2b, compared to the wild-type virus.

Conclusion

Therapies combining at least two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents may be effective for HCV-infected patients with NS5A-P32del.

Keywords

Direct-acting antiviral drug (DAA) Resistance-associated substitutions (RASs) Recombinant HCV Humanized liver mice Thymidine kinase transgene (TK-NOG) mice 

Abbreviations

HCV

Hepatitis C virus

DAA

Direct-acting antiviral drug

RASs

Resistance-associated substitutions

VF

Virologic failure

P32del

NS5A-P32 deletion mutant

TK-NOG

Thymidine kinase transgene

SVR

Sustained viral response

Notes

Acknowledgments

We would like to thank Gilead Sciences Inc. (Foster City, CA, USA) for providing ledipasvir and GS-558093. This work was partly supported by a Grant-in-Aid for Research on Hepatitis from the Japanese Agency for Medical Research and Development (JP18fk0210021, JP18fk0210002, JP18fk0210025), Ministry of Education, Culture, Sports, Science and Technology-Japan (JP16H05285), and Japan Society for the Promotion of Science (JP17K19647).

Disclosure

Dr. Tetsuo Takehara has received grants from Chugai Pharmaceutical Co. Ltd., Bristol-Myers Squibb, MSD K.K., AbbVie Inc. and Gilead Sciences. Dr. Tetsuo Takehara is on the speakers’ bureau for Bristol-Myers Squibb, MSD K.K., AbbVie Inc. and Gilead Sciences. Dr. Hayato Hikita has received grants from Bristol–Myers Squibb and MSD K.K. Dr. Ryotaro Sakamori and Dr. Tomohide Tatsumi have received grants from MSD K.K. Dr. Takahiro Kodama has received grants from MSD K.K. and Gilead Sciences. Dr. Yasuhiro Asahina belongs to a donation-funded department funded by Toray Industries Inc., Gilead Sciences, AbbVie GK, Chugai Pharmaceutical Co. Ltd., Fuji Rebio Inc., and Merck Sharp & Dohme.

Supplementary material

535_2018_1541_MOESM1_ESM.pptx (487 kb)
Supplementary material 1. Susceptibility of JFH1/5ACon1-wt, JFH1/5ACon1 with P32del (P32del), and JFH1/5ACon1 with L31M-P32del (L31M+P32del). Huh7.5.1 cells were electroporated with in vitro-transcribed HCV RNA. Four hours after electroporation, the culture media were replaced with fresh media containing NS5A inhibitors (A), NS3/4A inhibitors (B), NS5B inhibitors (C), and other inhibitors (D). After incubation for 72 hours, the cells were harvested, and the intracellular HCV core Ag levels were measured. The data are presented as percentages of the DMSO-treated control. Black circles indicate the HCV core Ag levels in JFH/5ACon1-wt. Red squares indicate the HCV core Ag levels in P32del. Blue diamonds indicate the HCV core Ag levels in L31M+P32del.

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Copyright information

© Japanese Society of Gastroenterology 2019

Authors and Affiliations

  • Akira Doi
    • 1
  • Hayato Hikita
    • 1
  • Yugo Kai
    • 1
  • Yuki Tahata
    • 1
  • Yoshinobu Saito
    • 1
  • Tasuku Nakabori
    • 1
  • Ryoko Yamada
    • 1
  • Takahiro Kodama
    • 1
  • Ryotaro Sakamori
    • 1
  • Asako Murayama
    • 2
  • Sayuri Nitta
    • 3
  • Yasuhiro Asahina
    • 3
    • 4
  • Hiroshi Suemizu
    • 5
  • Tomohide Tatsumi
    • 1
  • Takanobu Kato
    • 2
  • Tetsuo Takehara
    • 1
    Email author
  1. 1.Gastroenterology and Hepatology, Osaka University Graduate School of MedicineSuita, OsakaJapan
  2. 2.Department of Virology IINational Institute of Infectious DiseasesTokyoJapan
  3. 3.Department of Gastroenterology and HepatologyTokyo Medical and Dental UniversityTokyoJapan
  4. 4.Department of Liver Disease ControlTokyo Medical and Dental UniversityTokyoJapan
  5. 5.Department of Laboratory Animal ResearchCentral Institute for Experimental AnimalsKawasakiJapan

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