Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides

Abstract

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.

Keywords: ACE2; SARS-CoV-2; Spike; entry inhibitor.

Fig 1

ACE2-binding enhancing mutations, but not HR2, increase neutralization of SARS-CoV-2 Spike. (A) Schematic of different ACE2-Ig constructs. (B) Neutralization of VSV vectors pseudotyped with the Wuhan variant Spike protein by indicated amounts of different ACE2-Ig constructs. Error bars are SEM from n = 3 independent experiments, and mean IC₅₀ values are indicated. (C) Neutralization assay for VSV pseudovirus containing the D614G Spike variant. Error bars are SEM from n = 4 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 2

Spike-binding peptides attached to IgG1 Fc domain inhibit D614G pseudovirus. (A) Schematic of SBP-Ig and ACE2-V2.4-Ig constructs. (B) Neutralization assay for VSV pseudovirus containing the D614G Spike variant. Error bars are SEM from n = 4 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 3

Inhibition of entry mediated by different SARS-CoV-2 variants. Neutralization assays were performed with VSV pseudoviruses containing Spike proteins from the indicated SARS-CoV-2 variants: Alpha (A), Beta (B), Gamma (C), Delta (D), Epsilon (E), and Omicron BA.1 (F). In each case, pseudovirus entry was measured in the presence of the indicated inhibitors and doses. Error bars are SEM from n = 4 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 4

Importance of K417N/T and N501Y mutations in SBP-Ig resistance. Neutralization assays for VSV pseudoviruses containing indicated Spike proteins. D614G was used as a positive (sensitive) control and Beta variant B.1.351 as a negative (resistant) control. Single point mutations K417N, K417T, or E484K were introduced into D614G Spike and tested for sensitivity to (A) SBP1-Ig and (B) SBP2-Ig. (C) Point mutations N501Y and K417N + N501Y were introduced into D614G Spike and tested against SBP1-Ig. Error bars are SEM from n = 3 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 5

Effects of tandem combinations of SBP1 and SBP2 on neutralization of Spike variants. (A) Schematic of various SBP-Ig constructs. (B–G) Neutralization assays were performed against VSV pseudoviruses containing the indicated Spike proteins and treated with the indicated entry inhibitors and doses. Error bars are SEM from n = 3 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 6

Inhibition of replication-competent SARS-CoV-2. VeroE6-hACE2 cells were infected with 0.1 MOI of (A) Delta or (B) Omicron BA.1 SARS-CoV-2 virus, in the presence of various doses of the indicated inhibitors. After 2–3 days, cell viability was measured using an ATP luciferase assay to determine virus-induced cytotoxicity. Percent inhibition of virus replication was determined by comparing values normalized between infected and untreated control cells (0% inhibition value) and uninfected control cells (equivalent to 100% inhibition). Error bars are SEM from n = 3 independent experiments, and mean IC₅₀ values are indicated. P-values comparing the IC₅₀ between different inhibitor curves are shown in Table S1.

Fig 7

Protection of transgenic human ACE2 mice from SARS-CoV-2 Delta. Mice were infected intranasally with 10³ PFU of Delta variant and monitored daily for weight. Mice were euthanized if more than 20% body weight was lost, or health deteriorated. (A) Mice were injected IP with 10 µg ACE2-V2.4-Ig, at either 1 day (n = 16) or 1 plus 5 days (n = 5) post-infection, or were injected with PBS controls (n = 14). (B) Mice were injected IP at 12 h post-infection with 20 µg ACE2-V2.4-Ig (n = 5) or the molar equivalent for SBP1-1-Ig (7.9 µg) (n = 4), or a PBS control (n = 5). (C and D) Weights of individual mice over time from experiments (A and B).