SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

Fig 1. Upstream production of ACE2-Fc and ACE2(NN)-Fc using stable CHO cell lines.

(a) Representative IgG-Fc and ACE2 immunoblots using harvest media (15 μL) compared to blank media. CHO cell viable density (b) and viability (c) over a 7-day campaign using fed and unfed cultivation (n = 3). (d) Protein titer of the ACE2-Fc and ACE2(NN)-Fc during the campaigns under fed and unfed conditions (n = 3). (e) Representative IgG-Fc and ACE2 immunoblots using cell free harvest media (15 μL) collected on day 3, 5, and 7. Dots represent mean ± standard deviation.

Fig 2. Purity, identity, and activity characterization of purified ACE2-Fc and ACE2(NN)-Fc.

(a) Coomassie-stained SDS-PAGE gels under non reducing and reducing conditions of 1.5 μg of purified ACE2-Fc and ACE2(NN)-Fc proteins. (b) Anti-IgG-Fc and anti-ACE2 immunoblots of purified ACE2-fusion proteins using 0.5 ug of protein under non-reducing conditions. (c) ACE2 enzymatic activity of purified ACE2-Fc and ACE2(NN)-Fc proteins (n = 4). Two batch of ACE2-Fc and ACE2(NN)-Fc was performed in quadruplicate. Dots represent mean ± standard deviation and * indicates p <0.05.

Fig 3. ACE2-Fc and ACE2(NN)-Fc fusion proteins are heavily glycosylated with complex-type N-glycans.

Reduced SDS-page of ACE2-Fc (a) and ACE2(NN)-Fc (b) before and after treatment with PNGase F. (c) MALDI-TOF-MS spectra of permethylated N-glycans identified in ACE2-Fc (top) and ACE2(NN)-Fc(bottom). (d) Relative abundance of N-glycan peaks identified in ACE2-Fc and ACE2(NN)-Fc (Top), and comparison in percentage abundance of N-glycans identified in 3 batches of ACE2-Fc and 3 batches of ACE2(NN)-Fc (bottom).

Fig 4. ACE2-Fc and ACE2(NN)-Fc thermal stability assessment.

(a) Non-reduced Coomassie-stained SDS-page gels of the ACE2-Fc fusion proteins (1.5 μg) in the storage condition (-80°C) and at 4°C and 25°C for 1 and 7 days. Representative spectra of intrinsic fluorescent intensity curve changes of ACE-Fc (b) and ACE2(NN)-Fc (c) over a 20°C to 95°C temperature range at 1°C increments. The barycentric mean (BCM) of the intrinsic fluorescent curves was plotted against the temperature to determine onset of protein unfolding (T onset) and melt temperature (Tm) of ACE2-Fc (d) and ACE2(NN)-Fc (e). One batch of each ACE2 fusion protein was analyzed in quadruplicate (n = 4).

Fig 5. ACE2-Fc and ACE2(NN)-Fc binding affinity to S protein variants using biolayer interferometry.

Representative electropherograms of ACE2-Fc (a) and ACE2(NN)-Fc (b) binding to S protein variants: parental, alpha, beta, delta, and omicron. The binding affinity (c), disassociation rate (d), and association rate (e) of ACE2-Fc to S protein variants: parental, beta, alpha, delta, and omicron. Three batches of ACE2 fusion proteins were analyzed in triplicate (Two Way ANOVA, Factors: Variant and ACE2 fusion protein, n = 9). Dots represent the mean and bars represent the 95% Cl. * indicate p < 0.05 within the ACE2-Fc or ACE2(NN)-Fc groups.