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Viral infections are caused by the adhesion of viruses to host cell receptors, including sialylated glycans, glycosaminoglycans, and human blood group antigens (HBGAs). Atomic-level structural information on the interactions between viral particles or proteins with glycans can be determined to provide precise targets for designing antiviral drugs. Milk glycans, existing as free oligosaccharides or glycoconjugates, have attracted increasing attention; milk glycans protect infants against infectious diseases, particularly poorly manageable viral infections. Furthermore, several glycans containing structurally distinct sialic acid/fucose/sulfate modifications in human milk acting as a "receptor decoy" and serving as the natural antiviral library, could interrupt virus-receptor interaction in the first line of defense for viral infection. This review highlights the basis of virus-glycan interactions, presents specific glycan receptor binding by gastroenterovirus viruses, including norovirus, enteroviruses, and the breakthroughs in the studies on the antiviral properties of human milk glycans, and also elucidates the role of glycans in respiratory viruses infection. In addition, recent advances in methods for performing virus/viral protein-glycan interactions were reported. Finally, we discuss the prospects and challenges of the studies on the clinical application of human milk glycan for viral interventions.

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As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is an urgent need. Here, we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants, including BQ.1 and XBB.1, that are resistant to most clinically used monoclonal antibodies. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered the lung viral load by up to ~1,000-fold, prevented the emergence of clinical signs in >75% animals, and increased the animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously described ACE2-Ig constructs, we found that two constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the receptor binding domain (RBD)-binding interface should be avoided or performed with extra caution. Furthermore, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to the level of grams per liter, demonstrating the developability of them as biologic drug candidates. Stress condition stability testing of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. IMPORTANCE Engineering soluble ACE2 proteins that function as a receptor decoy to block SARS-CoV-2 infection is a very attractive approach to creating broadly effective and hard-to-escape anti-SARS-CoV-2 agents. This article describes development of two antibody-like soluble ACE2 proteins that broadly block diverse SARS-CoV-2 variants, including Omicron. In a stringent COVID-19 mouse model, both proteins successfully protected >87.5% animals from lethal SARS-CoV-2 infection. In addition, a head-to-head comparison of the two constructs developed in this study with five previously described ACE2 decoy constructs was performed here. Two previously described constructs with relatively more ACE2 surface mutations were found with less robust neutralization activities against diverse SARS-CoV-2 variants. Furthermore, the developability of the two proteins as biologic drug candidates was also assessed here. This study provides two broad anti-SARS-CoV-2 drug candidates and useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses.

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The Omicron variant has swept through most countries and become a dominant circulating strain, replacing the Delta variant. The evolutionary history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggests that the onset of another variant (possibly another variant of concern (VOC) is inevitable. Therefore, the development of therapeutics that enable treatments for all Omicron-included VOCs/variants of interest (VOIs) and future variants is desired. Recently, the recombinant receptor decoy therapeutic angiotensin-converting enzyme 2 (ACE2)-Fc has exhibited good safety in a phase 1 clinical trial; therefore, its variant-resistant profile needs to be understood. Here, we conducted a comprehensive evaluation of its neutralization breadth against the Omicron variant and other VOCs/VOIs. Furthermore, to evaluate its resistance to future variants, we investigated its ability to neutralize various single-residue mutated variants. Next, we demonstrated its resistance to evasion via an experiment that rapidly and effectively stimulates virus evolution with a replication-competent virus model. In addition, we evaluated its efficacy for cocktail therapy. The combination of ACE2-Fc and neutralizing antibodies showed both efficacy and breadth in the simulation experiment. The underlying mechanism was revealed to be a synergistic effect in the cocktails. Collectively, this study deepens the understanding of the resistance profile of recombinant receptor decoy therapeutics and highlights the potential value of ACE2-Fc and neutralizing antibody cocktails in the subsequent anti-SARS-CoV-2 campaign. Furthermore, we also provide an effective method to study the resistance profile of antiviral agents and rapidly screen for potential cocktails to combat future variants.

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The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here, we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351, and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralizing antibody approaches. Furthermore, we report a preclinical characterization package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralization capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralized four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilizing soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence, thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: while previous ACE2-based therapeutics have been described, ours has novel features, including (i) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system, (ii) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection, and reduced risk of hyperinflammation, and (iii) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralization potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic MAb.

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Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

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