Dimeric ACE2-FC is equivalent to monomeric ACE2 in the surrogate virus neutralization test

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Abstract

Angiotensin converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of the protective humoral immune response to a viral infection or vaccine using a surrogate virus neutralization test (sVNT). Soluble ACE2 is also being considered as an antiviral therapy option potentially insensitive to changes in the SARS-CoV-2 spike protein. For widespread sVHT testing, it is necessary to use ACE2 preparations or ACE2 conjugates with constant properties. Previously, we obtained a cell line that produces soluble monomeric ACE2 and showed that this variant of ACE2 can be used in sBHT, preferably in the form of a conjugate with horseradish peroxidase. To obtain a stable and universally applicable form of soluble ACE2, a cell line was obtained that produced the ACE2-Fc fusion protein with high productivity, more than 150 mg/l of the target protein during cultivation in a stirred flask. Affinity-purified ACE2-Fc is a mixture of dimeric and tetrameric forms, but allows one to obtain linearizable antibody inhibition curves for complexation with the receptor-binding domain of the SARS-CoV-2 spike protein. The ACE2-Fc-HRP based sVHT testing system can be used to practically measure the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

About the authors

D. E Kolesov

Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences

119071 Moscow, Russia

E. A Gaiamova

Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences

119071 Moscow, Russia

N. A Orlova

Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences

119071 Moscow, Russia

I. I Vorobiev

Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences

Email: ptichman@gmail.com
119071 Moscow, Russia

References

  1. Lambert, D. W., Yarski, M., Warner, F. J., Thornhill, P., Parkin, E. T., Smith, A. I., Hooper, N. M., and Turner, A. J. (2005) Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2), J. Biol. Chem., 280, 30113-30119, doi: 10.1074/jbc.M505111200.
  2. Smith, M. K., Tusell, S., Travanty, E. A., Berkhout, B., van der Hoek, L., and Holmes, K. V. (2006) Human angiotensin-converting enzyme 2 (ACE2) is a receptor for human respiratory coronavirus NL63, Adv. Exp. Med. Biol., 581, 285-288, doi: 10.1007/978-0-387-33012-9_48.
  3. Bohn, M. K., Lippi, G., Horvath, A., Sethi, S., Koch, D., Ferrari, M., Wang, C. B., Mancini, N., Steele, S., and Adeli, K. (2020) Molecular, serological, and biochemical diagnosis and monitoring of COVID-19: IFCC taskforce evaluation of the latest evidence, Clin. Chem. Lab. Med., 58, 1037-1052, doi: 10.1515/cclm-2020-0722.
  4. Bewley, K. R., Coombes, N. S., Gagnon, L., McInroy, L., Baker, N., Shaik, I., St-Jean, J. R., St-Amant, N., Buttigieg, K. R., Humphries, H. E., Godwin, K. J., Brunt, E., Allen, L., Leung, S., Brown, P. J., Penn, E. J., Thomas, K., Kulnis, G., Hallis, B., Carroll, M., et al. (2021) Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays, Nat. Protoc., 16, 3114-3140, doi: 10.1038/s41596-021-00536-y.
  5. Vandergaast, R., Carey, T., Reiter, S., Lathrum, C., Lech, P., Gnanadurai, C., Haselton, M., Buehler, J., Narjari, R., Schnebeck, L., Roesler, A., Sevola, K., Suksanpaisan, L., Bexon, A., Naik, S., Brunton, B., Weaver, S. C., Rafael, G., Tran, S., Baum, A., et al. (2021) IMMUNO-COV v2.0: Development and validation of a high-throughput clinical assay for measuring SARS-CoV-2-neutralizing antibody titers, mSphere, 6, e0017021, doi: 10.1128/mSphere.00170-21.
  6. Tan, C. W., Chia, W. N., Qin, X., Liu, P., Chen, M. I., Tiu, C., Hu, Z., Chen, V. C., Young, B. E., Sia, W. R., Tan, Y. J., Foo, R., Yi, Y., Lye, D. C., Anderson, D. E., and Wang, L. F. (2020) A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction, Nat. Biotechnol., 38, 1073-1078, doi: 10.1038/s41587-020-0631-z.
  7. Kolesov, D. E., Sinegubova, M. V., Dayanova, L. K., Dolzhikova, I. V., Vorobiev, I. I., and Orlova, N. A. (2022) Fast and accurate surrogate virus neutralization test based on antibody-mediated blocking of the interaction of ACE2 and SARS-CoV-2 spike protein RBD, Diagnostics (Basel), 12, 393, doi: 10.3390/diagnostics12020393.
  8. Рязанова А. Ю., Ходак Ю. А., Орлова Н. А., Синегубова М. В., Даянова Л. К., Ковнир С. В., Коробова С. В., Лёдов В. А., Ковальчук А. Л., Алхазова Б. И., Головина М. Э., Воробьёв И. И., Апарин П. Г. (2022) Рецепторсвязывающий домен S-белка SARS-CoV-2, слитый с негликозилированным кристаллизующимся фрагментом IgG1 человека: получение и оценка иммуногенности, Биотехнология, 38, 12-19, doi: 10.56304/S0234275822060102.
  9. Orlova, N. A., Dayanova, L. K., Gayamova, E. A., Sinegubova, M. V., Kovnir, S. V., and Vorobiev, I. I. (2022) Targeted knockout of the dhfr, glul, bak1, and bax genes by the multiplex genome editing in CHO cells, Dokl Biochem Biophys, 502, 40-44, doi: 10.1134/S1607672922010082.
  10. Matthews, A. M., Biel, T. G., Ortega-Rodriguez, U., Falkowski, V. M., Bush, X., Faison, T., Xie, H., Agarabi, C., Rao, V. A., and Ju, T. (2022) SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins, PLoS One, 17, e0278294, doi: 10.1371/journal.pone.0278294.
  11. Zhang, S., Gao, C., Das, T., Luo, S., Tang, H., Yao, X., Cho, C. Y., Lv, J., Maravillas, K., Jones, V., Chen, X., and Huang, R. (2022) The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies, J. Immunol. Methods, 503, 113244, doi: 10.1016/j.jim.2022.113244.
  12. Ru, Z., Xhang, Y., Wu, J., Huang, H., Liang, Y., Yang, X., Wu, J., and Lou, J. (2021) Comparison of the SARS-CoV-2 surrogate virus neutralization test (sVNT) assay and direct binding ELISA (S-IgG) with the cytopathic effect assay (CPE) in analyzing the neutralization antibody of vaccination people, J. Clin. Immunol. Immunother., 7, 063, doi: 10.24966/CIIT-8844/1000063.
  13. Pieri, M., Infantino, M., Manfredi, M., Nuccetelli, M., Grossi, V., Lari, B., Tomassetti, F., Sarubbi, S., Russo, E., Amedei, A., Benucci, M., Casprini, P., Stacchini, L., Castilletti, C., and Bernardini, S. (2022) Performance evaluation of four surrogate Virus Neutralization Tests (sVNTs) in comparison to the in vivo gold standard test, Front. Biosci. (Landmark Ed), 27, 74, doi: 10.31083/j.fbl2702074.
  14. Khan, A., Benthin, C., Zeno, B., Albertson, T. E., Boyd, J., Christie, J. D., Hall, R., Poirier, G., Ronco, J. J., Tidswell, M., Hardes, K., Powley, W. M., Wright, T. J., Siederer, S. K., Fairman, D. A., Lipson, D. A., Bayliffe, A. I., and Lazaar, A. L. (2017) A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome, Crit. Care, 21, 234, doi: 10.1186/s13054-017-1823-x.
  15. Shoemaker, R. H., Panettieri, R. A., Jr., Libutti, S. K., Hochster, H. S., Watts, N. R., Wingfield, P. T., Starkl, P., Pimenov, L., Gawish, R., Hladik, A., Knapp, S., Boring, D., White, J. M., Lawrence, Q., Boone, J., Marshall, J. D., Matthews, R. L., Cholewa, B. D., Richig, J. W., Chen, B. T., et al. (2022) Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer, PLoS One, 17, e0271066, doi: 10.1371/journal.pone.0271066.
  16. Chan, K. K., Dorosky, D., Sharma, P., Abbasi, S. A., Dye, J. M., Kranz, D. M., Herbert, A. S., and Procko, E. (2020) Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2, Science, 369, 1261-1265, doi: 10.1126/science.abc0870.
  17. Wines, B. D., Kurtovic, L., Trist, H. M., Esparon, S., Lopez, E., Chappin, K., Chan, L. J., Mordant, F. L., Lee, W. S., Gherardin, N. A., Patel, S. K., Hartley, G. E., Pymm, P., Cooney, J. P., Beeson, J. G., Godfrey, D. I., Burrell, L. M., van Zelm, M. C., Wheatley, A. K., Chung, A. W., et al. (2022) Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2, Front. Immunol., 13, 889372, doi: 10.3389/fimmu.2022.889372.
  18. Higuchi, Y., Suzuki, T., Arimori, T., Ikemura, N., Mihara, E., Kirita, Y., Ohgitani, E., Mazda, O., Motooka, D., Nakamura, S., Sakai, Y., Itoh, Y., Sugihara, F., Matsuura, Y., Matoba, S., Okamoto, T., Takagi, J., and Hoshino, A. (2021) Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2, Nat. Commun., 12, 3802, doi: 10.1038/s41467-021-24013-y.
  19. Moore, M. J., Dorfman, T., Li, W., Wong, S. K., Li, Y., Kuhn, J. H., Coderre, J., Vasilieva, N., Han, Z., Greenough, T. C., Farzan, M., and Choe, H. (2004) Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2, J. Virol., 78, 10628-10635, doi: 10.1128/JVI.78.19.10628-10635.2004.
  20. Svilenov, H. L., Delhommel, F., Siebenmorgen, T., Ruhrnossl, F., Popowicz, G. M., Reiter, A., Sattler, M., Brockmeyer, C., and Buchner, J. (2023) Extrinsic stabilization of antiviral ACE2-Fc fusion proteins targeting SARS-CoV-2, Commun. Biol., 6, 386, doi: 10.1038/s42003-023-04762-w.
  21. Hernández, T., Bermúdez, E., Fundora, T., and Sánchez, B. (2022) COVID-19 therapy based on soluble ACE2: the use of receptor-Fc fusion proteins, Open Acc. J. Bio Sci., 4, 1970-1975, doi: 10.38125/OAJBS.000472.

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