Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Analysis of SARS-CoV-2 Mutations after Nirmatrelvir Treatment in a Lung Cancer Xenograft Mouse Model

  • Bo Min Kang (Department of Microbiology, College of Medicine, Hallym University) ;
  • Dongbum Kim (Institute of Medical Science, College of Medicine, Hallym University) ;
  • Jinsoo Kim (Institute of Medical Science, College of Medicine, Hallym University) ;
  • Kyeongbin Baek (Department of Microbiology, College of Medicine, Hallym University) ;
  • Sangkyu Park (Department of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Ha-Eun Shin (Department of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Myeong-Heon Lee (Department of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Minyoung Kim (Department of Microbiology, College of Medicine, Hallym University) ;
  • Suyeon Kim (Department of Microbiology, College of Medicine, Hallym University) ;
  • Younghee Lee (Department of Biochemistry, College of Natural Sciences, Chungbuk National University) ;
  • Hyung-Joo Kwon (Department of Microbiology, College of Medicine, Hallym University)
  • Received : 2023.11.06
  • Accepted : 2023.12.18
  • Published : 2024.07.01
Download PDF
⟨ Previous Next ⟩

Abstract

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (Mpro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in Mpro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the Mpro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC50 values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

Keywords

Acknowledgement

We thank the National Culture Collection for Pathogens (Osong, Korea) for supplying parental SARS-CoV-2 and SARS-CoV-2 Omicron. This research was supported by grants from the National Research Foundation (NRF-2022M3A9I2082292) funded by the Ministry of Science and ICT in the Republic of Korea and by a grant of the Korea Health Technology R&D Project (grant number: HV23C0052) through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare in the Republic of Korea.

References

  1. Babraham Institute (2023) Babraham Bioinformatics. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ [accessed 2023 Feb 7].
  2. Chandrashekar, A., Liu, J., Martinot, A. J., McMahan, K., Mercado, N. B., Peter, L., Tostanoski, L. H., Yu, J., Maliga, Z., Nekorchuk, M., Busman-Sahay, K., Terry, M., Wrijil, L. M., Ducat, S., Martinez, D. R., Atyeo, C., Fischinger, S., Burke, J. S., Slein, M. D., Pessaint, L., Van Ry, A., Greenhouse, J., Taylor, T., Blade, K., Cook, A., Finneyfrock, B., Brown, R., Teow, E., Velasco, J., Zahn, R., Wegmann, F., Abbink, P., Bondzie, E. A., Dagotto, G., Gebre, M. S., He, X., Jacob-Dolan, C., Kordana, N., Li, Z., Lifton, M. A., Mahrokhian, S. H., Maxfield, L. F., Nityanandam, R., Nkolola, J. P., Schmidt, A. G., Miller, A. D., Baric, R. S., Alter, G., Sorger, P. K., Estes, J. D., Andersen, H., Lewis, M. G. and Barouch, D. H. (2020) SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science 369, 812-817. https://doi.org/10.1126/science.abc4776
  3. Danecek, P., Schiffels, S. and Durbin, R. (2016) Multiallelic Calling Model in Bcftools (-m). Available from: http://samtools.github.io/bcftools/call-m.pdf/ [accessed 2023 Feb 7].
  4. Deng, X., StJohn, S. E., Osswald, H. L., O'Brien, A., Banach, B. S., Sleeman, K., Ghosh, A. K., Mesecar, A. D. and Baker, S. C. (2014) Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance. J. Virol. 88, 11886-11898. https://doi.org/10.1128/JVI.01528-14
  5. Donovan-Banfield, I., Penrice-Randal, R., Goldswain, H., Rzeszutek, A. M., Pilgrim, J., Bullock, K., Saunders, G., Northey, J., Dong, X., Ryan, Y., Reynolds, H., Tetlow, M., Walker, L. E., FitzGerald, R., Hale, C., Lyon, R., Woods, C., Ahmad, S., Hadjiyiannakis, D., Periselneris, J., Knox, E., Middleton, C., Lavelle-Langham, L., Shaw, V., Greenhalf, W., Edwards, T., Lalloo, D. G., Edwards, C. J., Darby, A. C., Carroll, M. W., Griffiths, G., Khoo, S. H., Hiscox, J. A. and Fletcher, T. (2022) Characterisation of SARS-CoV-2 genomic variation in response to molnupiravir treatment in the AGILE Phase IIa clinical trial. Nat. Commun. 13, 7284. https://doi.org/10.1038/s41467-022-34839-9
  6. FDA (2023) FDA Approves First Oral Antiviral for Treatment of COVID-19 in Adults. Available from: https://www.fda.gov/news-events/press-announcements/fda-approves-first-oral-antiviral-treatment-covid-19-adults/[accessed 2023 Oct 10].
  7. Guo, K., Barrett, B. S., Morrison, J. H., Mickens, K. L., Vladar, E. K., Hasenkrug, K. J., Poeschla, E. M. and Santiago, M. L. (2022) Interferon resistance of emerging SARS-CoV-2 variants. Proc. Natl. Acad. Sci. U. S. A. 119, e2203760119.
  8. Hammond, J., Leister-Tebbe, H., Gardner, A., Abreu, P., Bao, W., Wisemandle, W., Baniecki, M., Hendrick, V. M., Damle, B., Simon-Campos, A., Pypstra, R., Rusnak, J. M. and EPIC-HR Investigators. (2022) Oral nirmatrelvir for high-risk, nonhospitalized adults with Covid-19. N. Engl. J. Med. 386, 1397-1408. https://doi.org/10.1056/NEJMoa2118542
  9. Iketani, S., Liu, L., Guo, Y., Liu, L., Chan, J. F., Huang, Y., Wang, M., Luo, Y., Yu, J., Chu, H., Chik, K. K., Yuen, T. T., Yin, M. T., Sobieszczyk, M. E., Huang, Y., Yuen, K. Y., Wang, H. H., Sheng, Z. and Ho, D. D. (2022) Antibody evasion properties of SARS-CoV-2 Omicron sublineages. Nature 604, 553-556. https://doi.org/10.1038/s41586-022-04594-4
  10. Iketani, S., Mohri, H., Culbertson, B., Hong, S. J., Duan, Y., Luck, M. I., Annavajhala, M. K., Guo, Y., Sheng, Z., Uhlemann, A. C., Goff, S. P., Sabo, Y., Yang, H., Chavez, A. and Ho, D. D. (2023) Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir. Nature 613, 558-564. https://doi.org/10.1038/s41586-022-05514-2
  11. Jochmans, D., Liu, C., Donckers, K., Stoycheva, A., Boland, S., Stevens, S. K., De Vita, C., Vanmechelen, B., Maes, P., Trueb, B., Ebert, N., Thiel, V., De Jonghe, S., Vangeel, L., Bardiot, D., Jekle, A., Blatt, L. M., Beigelman, L., Symons, J. A., Raboisson, P., Chaltin, P., Marchand, A., Neyts, J., Deval, J. and Vandyck, K. (2023) The substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir. mBio 14, e0281522. https://doi.org/10.1128/mbio.02815-22
  12. Kim, D., Kim, J., An, S., Kim, M., Baek, K., Kang, B. M., Maharjan, S., Kim, S., Hwang, S. Y., Park, I. G., Park, S., Suh, J. G., Park, M. S., Noh, M., Lee, Y. and Kwon, H. J. (2023a) In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein. J. Med. Virol. 95, e28626.
  13. Kim, D., Kim, J., Kim, M., Park, H., Park, S., Maharjan, S., Baek, K., Kang, B. M., Kim, S., Park, M. S., Lee, Y. and Kwon, H. J. (2023b) Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2. Front. Cell. Infect. Microbiol. 13, 1280686.
  14. Kim, D., Kim, J., Park, S., Kim, M., Baek, K., Kang, M., Choi, J. K., Maharjan, S., Akauliya, M., Lee, Y. and Kwon, H. J. (2021a) Production of SARS-CoV-2 N protein-specific monoclonal antibody and its application in an ELISA-based detection system and targeting the interaction between the spike C-terminal domain and N protein. Front. Microbiol. 12, 726231.
  15. Kim, D., Maharjan, S., Kim, J., Park, S., Park, J. A., Park, B. K., Lee, Y. and Kwon, H. J. (2021b) MUC1-C influences cell survival in lung adenocarcinoma Calu-3 cells after SARS-CoV-2 infection. BMB Rep. 54, 425-430. https://doi.org/10.5483/BMBRep.2021.54.8.018
  16. Kim, J., Hwang, S. Y., Kim, D., Kim, M., Baek, K., Kang, M., An, S., Gong, J., Park, S., Kandeel, M., Lee, Y., Noh, M. and Kwon, H. J. (2022) abiraterone acetate attenuates SARS-CoV-2 replication by interfering with the structural nucleocapsid protein. Biomol. Ther. (Seoul) 30, 427-434. https://doi.org/10.4062/biomolther.2022.037
  17. Lei, C., Yang, J., Hu, J. and Sun, X. (2021) On the calculation of TCID50 for quantitation of virus infectivity. Virol. Sin. 36, 141-144. https://doi.org/10.1007/s12250-020-00230-5
  18. Moghadasi, S. A., Heilmann, E., Khalil, A. M., Nnabuife, C., Kearns, F. L., Ye, C., Moraes, S. N., Costacurta, F., Esler, M. A., Aihara, H., von Laer, D., Martinez-Sobrido, L., Palzkill, T., Amaro, R. E. and Harris, R. S. (2023) Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors. Sci. Adv. 9, eade8778. https://doi.org/10.1126/sciadv.ade8778
  19. Munster, V. J, Feldmann, F., Williamson, B. N., van Doremalen, N., Perez-Perez, L., Schulz, J., Meade-White, K., Okumura, A., Callison, J., Brumbaugh, B., Avanzato, V. A., Rosenke, R., Hanley, P. W., Saturday, G., Scott, D., Fischer, E. R. and de Wit, E. (2020) Respiratory disease in rhesus macaques inoculated with SARS-CoV-2. Nature 585, 268-272. https://doi.org/10.1038/s41586-020-2324-7
  20. Owen, D. R., Allerton, C. M. N., Anderson, A. S., Aschenbrenner, L., Avery, M., Berritt, S., Boras, B., Cardin, R. D., Carlo, A., Coffman, K. J., Dantonio, A., Di, L., Eng, H., Ferre, R., Gajiwala, K. S., Gibson, S. A., Greasley, S. E., Hurst, B. L., Kadar, E. P., Kalgutkar, A. S., Lee, J. C., Lee, J., Liu, W., Mason, S. W., Noell, S., Novak, J. J., Obach, R. S., Ogilvie, K., Patel, N. C., Pettersson, M., Rai, D. K., Reese, M. R., Sammons, M. F., Sathish, J. G., Singh, R. S. P., Steppan, C. M., Stewart, A. E., Tuttle, J. B., Updyke, L., Verhoest, P. R., Wei, L., Yang, Q. and Zhu, Y. (2021) An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586-1593. https://doi.org/10.1126/science.abl4784
  21. Park, S., Kim, D., Kim, J., Kwon, H. J. and Lee, Y. (2023) SARS-CoV-2 infection induces expression and secretion of lipocalin-2 and regulates iron in a human lung cancer xenograft model. BMB Rep. 56, 669-674. https://doi.org/10.5483/BMBRep.2023-0177
  22. Park, B. K., Kim, D., Park, S., Maharjan, S., Kim, J., Choi, J. K., Akauliya, M., Lee, Y. and Kwon, H. J. (2021) Differential signaling and virus production in Calu-3 cells and Vero cells upon SARS-CoV-2 infection. Biomol. Ther. (Seoul) 29, 273-281. https://doi.org/10.4062/biomolther.2020.226
  23. Rockx, B., Kuiken, T., Herfst, S., Bestebroer, T., Lamers, M. M., Oude Munnink, B. B., de Meulder, D., van Amerongen, G., van den Brand, J., Okba, N. M. A., Schipper, D., van Run, P., Leijten, L., Sikkema, R., Verschoor, E., Verstrepen, B., Bogers, W., Langermans, J., Drosten, C., Fentener van Vlissingen, M., Fouchier, R., de Swart, R., Koopmans, M. and Haagmans, B. L. (2020) Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model. Science 368, 1012-1015. https://doi.org/10.1126/science.abb7314
  24. Sefik, E., Israelow, B., Mirza, H., Zhao, J., Qu, R., Kaffe, E., Song, E., Halene, S., Meffre, E., Kluger, Y., Nussenzweig, M., Wilen, C. B., Iwasaki, A. and Flavell, R. A. (2022) A humanized mouse model of chronic COVID-19. Nat. Biotechnol. 40, 906-920. https://doi.org/10.1038/s41587-021-01155-4
  25. Szemiel, A. M., Merits, A., Orton, R. J., MacLean, O. A., Pinto, R. M., Wickenhagen, A., Lieber, G., Turnbull, M. L., Wang, S., Furnon, W., Suarez, N. M., Mair, D., da Silva, Filipe, A., Willett, B. J., Wilson, S. J., Patel, A. H., Thomson, E. C., Palmarini, M., Kohl, A. and Stewart, M. E. (2021) In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. PLoS Pathog. 17, e1009929.
  26. Tan, B., Joyce, R., Tan, H., Hu, Y. and Wang, J. (2023) SARS-CoV-2 main protease drug design, assay development, and drug resistance studies. Acc. Chem. Res. 56, 157-168. https://doi.org/10.1021/acs.accounts.2c00735
  27. Wang, Q., Guo, Y., Iketani, S., Nair, M. S., Li, Z., Mohri, H., Wang, M., Yu, J., Bowen, A. D., Chang, J. Y., Shah, J. G., Nguyen, N., Chen, Z., Meyers, K., Yin, M. T., Sobieszczyk, M. E., Sheng, Z., Huang, Y., Liu, L. and Ho, D. D. (2022) Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4 and BA.5. Nature 608, 603-608. https://doi.org/10.1038/s41586-022-05053-w
  28. Wu, G., Kim, D., Kim, J. N., Park, S., Maharjan, S., Koh, H., Moon, K., Lee, Y. and Kwon, H. J. (2018) A mucin1 C-terminal subunit-directed monoclonal antibody targets overexpressed mucin1 in breast cancer. Theranostics 8, 78-91. https://doi.org/10.7150/thno.21278
  29. Xiong, M., Su, H., Zhao, W., Xie, H., Shao, Q. and Xu, Y. (2021) What coronavirus 3C-like protease tells us: from structure, substrate selectivity, to inhibitor design. Med. Res. Rev. 41, 1965-1998. https://doi.org/10.1002/med.21783
  30. Zhou, Y., Gammeltoft, K. A., Ryberg, L. A., Pham, L. V., Tjornelund, H. D., Binderup, A., Duarte, Hernandez, C. R., Fernandez-Antunez, C., Offersgaard, A., Fahnoe, U., Peters, G. H. J., Ramirez, S., Bukh, J. and Gottwein, J. M. (2022) Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system. Sci. Adv. 8, eadd7197. https://doi.org/10.1126/sciadv.add7197
  31. Zhou, Y., Zhi, H. and Teng, Y. (2023) The outbreak of SARS-CoV-2 Omicron lineages, immune escape, and vaccine effectivity. J. Med. Virol. 95, e28138.