High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses.

The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron BA.1 and Omicron BA.2 now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in a recombinant adeno-associated virus vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron BA.1 and Omicron BA.2. Tight decoy binding tracks with human ACE2 binding of viral spike receptor-binding domains across diverse clades of coronaviruses. Furthermore, in a coronavirus that cannot bind human ACE2, a variant that acquired human ACE2 binding was bound by the decoy with nanomolar affinity. Considering these results, we discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats.

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

Fluorescent Sensors That Enable a General Method To Quantify Affinities of Receptor Proteins for Polyubiquitin Ligands.

In all eukaryotic cells, modifications of proteins by polymers of ubiquitin (polyUb) are signals used in diverse biological processes. To better understand how polyUb signals are read and promote their different functions, quantitative measurements of their interactions with receptor proteins are needed. However, affinities and selectivities of different forms of polyUb with various receptors have been difficult to determine because the availability of well-defined polyUb chains can be limiting and there is a lack of general, sensitive methods to assay their interactions. We have addressed this challenge by developing a series of fluorescent protein sensors for polyUb; by competition of the sensors against receptor proteins in vitro for limiting amounts of polyUb, receptor·polyUb affinities can be quantified. Due to the high affinities of the polyUb sensors (Kd ∼ 10-9 M), binding assays using this competition format require much less polyUb (<0.1%) than would be needed in direct titrations of the polyUb ligands. Furthermore, the high sensitivity and large dynamic range of the sensor fluorescence readout allow for precise measurements even for very tight interactions (i.e., nanomolar Kd). Importantly, as demonstrated here with Ub2 and Ub3 ligands, the assay does not require labeling of either the receptor protein or the polyUb, and it can be used with polyUb ligands composed of virtually any Ub-Ub linkage type.