The ongoing SARS-CoV-2 pandemic highlights the virus's capacity for rapid mutation, leading to immune escape and reduced efficacy of vaccines and monoclonal antibodies. The emergence of Omicron subvariants, particularly BA.5, underscores this challenge, demonstrating substantial immune evasion compared to earlier variants. While monoclonal antibodies and vaccination have been vital tools in the fight against SARS-CoV-2, the effectiveness of these approaches against Omicron subvariants is waning, necessitating the development of updated boosters and novel therapeutics. The need for pan-coronavirus therapeutics resistant to mutational escape is paramount as the virus transitions towards an endemic state. Engineered ACE2 decoys offer a promising strategy to combat SARS-CoV-2 infection due to their ability to outcompete native ACE2 receptors and neutralize the virus. Their structural similarity to the native ACE2 receptor enables them to maintain effectiveness against evolving viral variants. However, a critical balance must be struck between achieving tight binding affinity and maintaining broad neutralization capabilities. Overly engineered ACE2 decoys might compromise their ability to neutralize a broad range of SARS-CoV-2 variants. This research focuses on evaluating the efficacy of a computationally designed ACE2 decoy, termed FLIF, which demonstrates picomolar affinity for the Delta variant and robust neutralization of Omicron subvariants. The study aims to address the crucial question of whether engineered ACE2 decoys maintain their efficacy against the latest circulating Omicron subvariants, particularly BA.5, which carries unique mutations (F486V and L452R) not present in earlier Omicron variants and known to contribute to antibody escape. This in-depth investigation provides critical insights into the potential of affinity-enhanced ACE2 decoys in combating evolving SARS-CoV-2 variants.

Several research groups have pursued the engineering of ACE2 decoys using various strategies. Some have combined computational design with experimental approaches like random mutagenesis and selection using yeast surface display, while others have relied solely on experimental methods. A substantial body of work uses purely computational approaches. While several computationally designed ACE2 decoys have been tested in vitro, in vivo testing, especially against newer Omicron subvariants such as BA.5, has been limited. To date, no computationally or experimentally engineered ACE2 decoy has been thoroughly tested in vivo against Omicron BA.5. Prior studies explored the in vitro and in vivo efficacy of affinity-matured ACE2 decoys against earlier variants, such as BA.1, but direct comparisons with soluble wild-type ACE2 against newer Omicron variants have been lacking. Thus, this study aims to fill the gap by providing a comprehensive in vitro and in vivo evaluation of FLIF, a computationally designed ACE2 decoy, against Omicron BA.5.

The study employed a multi-faceted approach, combining computational protein design, molecular dynamics (MD) simulations, free energy calculations, and experimental validation. **Computational Design:** FLIF was designed using an orthogonal approach, integrating computational protein design, MD simulations, and free energy calculations. Rosetta protein design software was used to introduce mutations in ACE2 (T27F, Q42L, L79I, N330F) based on previous designs. Further refinement was done using the Rosetta "Coupled Moves" protocol, focusing on the local environment around the introduced mutations. The resulting designs were evaluated based on cross-interface pairwise interactions between RBD and ACE2, and the top designs were selected for further analysis. **Molecular Dynamics Simulations:** MD simulations were performed using the AMBER 20 package to calculate binding enthalpy and provide insights into affinity enhancement. The simulations involved wild-type ACE2 and the FLIF mutant interacting with BA.4/5 RBD. Volumetric maps were created to visualize the 3D space occupied by key residues in the RBD and interacting ACE2 residues. Analysis of hydrogen bond networks and native contacts of key ACE2 residues with the BA.4/5 RBD provided insights into the improved binding of FLIF. **Free Energy Calculations:** MM/GBSA and CL-FEP approaches were used to calculate relative and absolute binding free energies, respectively. The MM/GBSA calculations utilized snapshots from 100 ns MD simulations, while CL-FEP employed 300 ns MD simulations of individual proteins (RBD, ACE2, RBD-ACE2 complex, and bulk solvent) to assess absolute binding free energies. **Experimental Validation:** The study involved several experimental assays to validate the computational findings. Flow cytometry assessed the binding of sACE2-IgG1 fusion proteins to BA.2 Omicron spike proteins expressed on the surface of Expi293F cells. Biolayer interferometry (BLI) measured the binding kinetics of sACE2-IgG1 proteins to delta RBD-8h. Pseudovirus neutralization assays were conducted to evaluate the neutralization of spike protein-expressing pseudoviruses by sACE2-IgG1 proteins. Finally, live SARS-CoV-2 Omicron BA.5 neutralization and in vivo hamster infection studies were used to assess the therapeutic efficacy of FLIF against authentic virus.

The study's key findings demonstrated that FLIF, the engineered ACE2 decoy, exhibits enhanced binding affinity and neutralization capacity compared to wild-type ACE2 against various SARS-CoV-2 variants, including Omicron BA.5. **Computational Findings:** MD simulations showed that FLIF forms stronger hydrophobic interactions and a strengthened hydrogen bond network with the BA.4/5 RBD, compared to wild-type ACE2. The volumetric maps highlighted alterations in the orientation of key residues in the RBD and ACE2 upon mutation, leading to improved shape complementarity in the FLIF-RBD interface. MM/GBSA and CL-FEP calculations confirmed significantly higher binding affinities for FLIF to BA.4/5 RBD compared to wild-type ACE2. **Experimental Findings:** Flow cytometry and BLI assays confirmed strong binding of FLIF to SARS-CoV-2 variants. Pseudovirus neutralization assays showed that FLIF effectively neutralizes SARS-CoV-2 variants including BA.1, BA.2, and BA.4/5. Notably, in vivo studies in Syrian hamsters challenged with Omicron BA.5 demonstrated significant reduction in viral load in the lungs of FLIF-treated animals compared to the control group. FLIF exhibited therapeutic efficacy when administered 2 hours post-infection. Overall, the data consistently demonstrates FLIF's enhanced binding and potent neutralization against a range of SARS-CoV-2 variants.

The findings of this study highlight the successful design and validation of FLIF, an engineered ACE2 decoy, as a potent therapeutic against SARS-CoV-2, including the Omicron BA.5 variant. The orthogonal approach, combining computational design and experimental validation, proved successful in developing an ACE2 decoy with superior binding affinity and broad neutralization capability. The improved binding affinity is attributed to strengthened hydrophobic interactions and the hydrogen bond network resulting from specific mutations in FLIF. The in vivo efficacy observed in hamsters further supports the potential of FLIF as a therapeutic agent. The enhanced performance of FLIF compared to wild-type sACE2 underscores the advantage of affinity-enhanced ACE2 decoys against evolving SARS-CoV-2 variants. While the study has demonstrated success, the limitations of attenuated disease in the hamster model and the post-infection treatment timing need to be considered in future work.

This study successfully designed and validated FLIF, an engineered ACE2 decoy, as a highly potent neutralizer against a wide range of SARS-CoV-2 variants, including Omicron BA.5, both in vitro and in vivo. The orthogonal combination of computational design and experimental validation proved extremely effective in developing a therapeutic candidate with superior binding and neutralization capabilities. The results strongly suggest that affinity-enhanced ACE2 decoys like FLIF might be necessary to effectively combat the evolving threats posed by SARS-CoV-2 variants. Future studies should focus on optimizing FLIF for broader clinical applications and investigating its efficacy against other emerging SARS-related coronaviruses.

The in vivo studies utilized a hamster model, where Omicron variants cause attenuated disease, limiting the assessment of FLIF's effect on severe lung pathology observed with more virulent variants. The 2-hour post-infection treatment timing may not fully reflect the clinical scenario in humans. While previous studies indicated therapeutic efficacy of other ACE2 decoys with later administration, further investigations with FLIF under different treatment timelines are warranted.