Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test

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Denis E. Kolesov¹, Elizaveta A. Gaiamova¹, Nadezhda A. Orlova¹, and Ivan I. Vorobiev^1,a*

¹Federal Research Center of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia

^* To whom correspondence should be addressed.

Received May 13, 2023; Revised June 14, 2023; Accepted June 16, 2023

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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 protective humoral immune response to a viral infection or vaccine using the surrogate virus neutralization test (sVNT). Soluble ACE2 is also considered as an option for antiviral therapy potentially insensitive to the changes in the SARS-CoV-2 spike protein. Extensive testing of the samples of patient’s serum by the sVNT method requires using preparations of ACE2 or ACE2 conjugates with constant properties. We have previously obtained a cell line that is a producer of a soluble monomeric ACE2 and showed that this ACE2 variant can be used in sVNT, preferably as a conjugate with horseradish peroxidase. A cell line that generates an ACE2-Fc fusion protein with high productivity, more than 150 mg/liter of the target protein when cultured in a stirred flask, was obtained for producing a stable and universally applicable form of soluble ACE2. The affinity-purified ACE2-Fc fusion contains a mixture of dimeric and tetrameric forms, but allows obtaining linear response curves for inhibition of binding with the receptor-binding domain of the SARS-CoV-2 spike protein by antibodies. The ACE2-Fc-HRP-based sVNT testing system can be used for practical measurements of the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

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KEY WORDS: angiotensin-converting enzyme 2, SARS-CoV-2, sVNT, virus neutralization, protective immunity

DOI: 10.1134/S0006297923090079

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