Redirection of SARS-CoV-2 to phagocytes by intranasal sACE2-Fc as a universal decoy confers complete prophylactic protection

Reviewer #1 (Public review):

Summary:

This manuscript by Wang et al. describes the development of an optimized soluble ACE2-Fc fusion protein, B5-D3, for intranasal prophylaxis against SARS-CoV-2. As shown, B5-D3 conferred protection not only by acting as a neutralizing decoy, but also by redirecting virus-decoy complexes to phagocytic cells for lysosomal degradation. The authors showed complete in vivo protection in K18-hACE2 mice and investigated the underlying mechanism by a combination of Fc-mutant controls, transcriptomics, biodistribution studies, and in vitro assays.

Strengths:

The major strength of this work is the identification of a novel antiviral approach with broad-spectrum and beyond simple neutralization. Mutant ACE2 enables broad and potent binding activity with the S proteins of SARS-CoV-2 variants, while the fused Fc part mediates phagocytosis to clear the viral particles. The conceptual advance of this ACE2-Fc combination is convincingly validated by in vivo protection data and by the completely abrogated protection of Fc LALA mutant.

Weaknesses:

Some aspects could be further modified.

(1) A previously reported ACE2 decamer (DOI: 10.1080/22221751.2023.2275598) needs to be mentioned and compared in the Discussion part.

(2) Limitations of this study, such as off-target binding and potential immunogenicity, should also be discussed.

Reviewer #2 (Public review):

Summary:

Wang et al. engineered an optimized ACE2 mutant by introducing two mutations (T92Q and H374N) and fused this ACE2 mutant to human IgG1-Fc (B5-D3). Experimental results suggest that B5-D3 exhibits broad-spectrum neutralization capacity and confers effective protection upon intranasal administration in SARS-CoV-2-infected K18-hACE2 mice. Transcriptomic analysis suggests that B5-D3 induces early immune activation in lung tissues of infected mice. Fluorescence-based bio-distribution assay further indicates rapid accumulation of B5-D3 in the respiratory tract, particularly in airway macrophages. Further investigation shows that B5-D3 promotes viral phagocytic clearance by macrophages via an Fc-mediated effector function, namely antibody-dependent cellular phagocytosis (ADCP), while simultaneously blocking ACE2-mediated viral infection in epithelial cells. These results provide insights into improving decoy treatments against SARS-CoV-2 and other potential respiratory viruses.

Strengths:

The protective effect of this ACE2-Fc fusion protein against SARS-CoV-2 infection has been evaluated in a quite comprehensive way.

Weaknesses:

(1) The paper lacks an explanation regarding the reason for the combination of mutations listed in Supplementary Figure 2b. For example, for the mutations that enhance spike protein binding, B2-B6 does not fully align with the mutations listed in Table S1 of Reference 4, yet no specific criteria are provided. Second, for the mutations that abolished enzymatic activity, while D1 and D2, D3, D4, and D5 are cited from References 12, 11, and 33, respectively, the reason for combining D3 and D4 into A2, and D1 and D2 into A3 remains unexplained. It is also unclear whether some of these other possible combinations have been tested. Furthermore, for the B5-derived mutations, only double-mutant combinations with D1-D5 are tested, with no attempt made to evaluate triple mutations involving A2 or A3.

(2) Figures 1b, 1d, and 1e lack statistical analyses, making it difficult to determine whether B5 and D3 exhibit significant advantages. For Wuhan-Hu-1 strain, B2 and B5 are similar, and for D614G strain, B2, B3, B4, B5, and B6 display comparable results. However, only the glycosylation-related single mutant B5 is chosen for further combinatorial constructs. Moreover, for VOC/VOI strains, B5 is superior to B5-D3; for the Alpha strain, B5-D4 and B5-D5 are superior to B5-D3; and for the Delta and Lambda strains, B5-D5 is superior to B5-D3. These observations further highlight the need for a clearer explanation of the selection strategy.

(3) Figure 1e does not specify the construct form of the control hIgG1, namely whether it is an hIgG1 Fc fragment or a full-length hIgG1 protein. If the full-length form is used, the design of its Fab region should be clarified to ensure the accuracy and comparability of the experimental control.

(4) In Figure 2a, all three PBS control mice died, whereas in Figure 2f, three out of five PBS control mice died, with the remaining showing gradual weight recovery. This discrepancy may reflect individual immune variations within the control groups, and it is necessary to clarify whether potential autoimmune factors could have affected the comparability of the results. Also, the mouse experiments suffer from insufficient sample sizes, which affects the statistical power and reliability of the results. In Figure 2a, each group contains only 4 replicates, one of which was used for lung tissue sampling. As a result, body weight monitoring data is derived from only 3 mice per group (the figure legend indicating n=4 should be corrected to n=3). Such a small sample size limits the robustness of the conclusions. Similarly, in Figure 2f, although each group has 5 replicates, body weight data are presented for only 4 mice, with no explanation provided for the exclusion of the fifth mouse. Furthermore, the lung tissue experiments in Figure 3a include only 3 replicates, which is also inadequate.

(5) Compared to 6 hours, intranasal administration of B5-D3 at 24 hours before viral infection results in reduced protective efficacy. However, only survival and body weight data are provided, with no supporting evidence from virological assays such as viral titer measurement. Therefore, the long-term effectiveness lacks sufficient experimental validation.

(6) In Figures 3b and 3c, viral spike (S) and nucleocapsid (N) RNA relative expression levels are quantified by qPCR. The results show significant individual variation within the B5-D3-LALA treatment group: one mouse exhibits high S and N expression, while the other two show low expression. Viral load levels are also inconsistent: two mice have high viral loads, and one has a low viral load. Due to this variability, the available data are insufficient to robustly support the conclusion.

(7) Figure 3e: "H&E staining indicated alveolar thickening in all groups," including the Mock group. Since the Mock group did not receive virus or active drug treatment, this observed change may result from local tissue reaction induced by the intranasal inoculation procedure itself, rather than specific immune activation. A control group (no manipulation) should be set to rule out potential confounding effects of the experimental procedure on tissue morphology, thereby allowing a more accurate assessment of the drug's effects.

(8) In Supplementary Figure 11b, a considerable number of alveolar macrophages (AMs) are observed in both the PBS and B5-D3 groups. This makes it difficult to determine whether the observed accumulation is specifically induced by B5-D3.

(9) In the flow cytometry experiment shown in Figure 5, the PBS control group is not labeled with AF750, which necessarily results in a value of zero for "B5-D3+ cells" on the y-axis. An appropriate control (e.g., hIgG1-Fc labeled with AF750) should be included.

(10) The Methods section: a more detailed description of the experimental procedures involving HIV p24 and SARS-CoV-2 should be included.

Reviewer #3 (Public review):

Strengths:

The core strength of this study lies in its innovative demonstration that an engineered sACE2-Fc fusion redirects virus-decoy complexes to Fc-mediated phagocytosis and lysosomal clearance in macrophages, revealing a distinct antiviral mechanism beyond traditional neutralization. Its complete prophylactic protection in animal models and precise targeting of airway phagocytes establish a novel therapeutic paradigm against SARS-CoV-2 variants and future respiratory viruses.

Weaknesses:

The study attributes the complete antiviral protection to Fc-mediated phagocytic clearance, a central claim that requires more rigorous experimental validation. The observation that abrogating Fc functions compromises protection could be confounded by potential alterations in the protein's stability, half-life, or overall structure. To firmly establish this mechanism, it is crucial to include a control molecule with a mutated Fc region that lacks FcγR binding while preserving the Fc structure itself. Without this critical control, the conclusion that phagocytic clearance is the primary mechanism remains inadequately supported. The strategy of deliberately targeting virus-decoy complexes to phagocytes via Fc receptors inherently raises the question of Antibody-Dependent Enhancement (ADE) of disease. While the authors demonstrate a lack of productive infection in macrophages, this only addresses one facet of ADE. The risk of Fc-mediated exacerbation of inflammation (ADE) remains a critical concern. The manuscript would be significantly strengthened by a direct discussion of this risk and by including data, such as cytokine profiling from treated macrophages, to more comprehensively address the safety profile of this approach. The exclusive use of the K18-hACE2 mouse model, which exhibits severe disease, limits the generalizability of the findings. The "complete protection" observed may not translate to models with more robust and naturalistic immune responses or to human physiology. Furthermore, the lack of data on circulating SARS-CoV-2 variants is a concern. The concept of sACE2-Fc fusion proteins as decoy receptors is not novel, and numerous similar constructs have been previously reported. The manuscript would benefit from a clearer demonstration of how the optimized B5-D3 mutant represents a significant advance over existing sACE2-Fc designs. A direct comparative analysis with previously published benchmarks, particularly in terms of neutralizing potency, Fc effector function strength, and in vivo efficacy, is necessary to establish the incremental value and novelty of this specific agent.