Antiviral Nanobiologic Therapy Remodulates Innate Immune Responses to Highly Pathogenic Coronavirus

Abstract

Highly pathogenic coronavirus (CoV) infection induces a defective innate antiviral immune response coupled with the dysregulated release of proinflammatory cytokines and finally results in acute respiratory distress syndrome (ARDS). A timely and appropriate triggering of innate antiviral response is crucial to inhibit viral replication and prevent ARDS. However, current medical countermeasures can rarely meet this urgent demand. Here, an antiviral nanobiologic named CoVR-MV is developed, which is polymerized of CoVs receptors based on a biomimetic membrane vesicle system. The designed CoVR-MV interferes with the viral infection by absorbing the viruses with maximized viral spike target interface, and mediates the clearance of the virus through its inherent interaction with macrophages. Furthermore, CoVR-MV coupled with the virus promotes a swift production and signaling of endogenous type I interferon via deregulating 7-dehydrocholesterol reductase (DHCR7) inhibition of interferon regulatory factor 3 (IRF3) activation in macrophages. These sequential processes re-modulate the innate immune responses to the virus, trigger spontaneous innate antiviral defenses, and rescue infected Syrian hamsters from ARDS caused by SARS-CoV-2 and all tested variants.

Keywords: ARDS; biomimetic nanocarrier; cell membrane vesicles; endogenous type I interferon; highly pathogenic coronavirus; imbalanced innate immune responses.

Figure 1

Imbalanced innate immune response and robust respiratory infection are typical features of highly pathogenic coronavirus‐caused ARDS. A) Schematic illustration for the characteristics of imbalanced innate immune responses after highly pathogenic coronavirus infection. Relative mRNA levels of B) IFN‐ α, IFN‐ β, MX1, and C) proinflammatory cytokines include IL‐6, TNF‐α and IFN‐γ in the lung tissues collected at 5 dpi, respectively (n = 5). The mRNA levels were standardized to the house‐keeping gene γ‐actin. D) Viral RNA levels in turbinate, trachea and lung tissues collected at 5 dpi were measured by RT‐PCR (n = 5). The primers of SARS‐CoV‐2 ORF1ab gene were used. E) Representative H&E staining images for lung lobes collected at 5 dpi were shown, Scale bar = 2 mm. F) Body weight changes of the hamsters from 0 to 5 dpi were recorded (n = 5). Data were shown as mean ± SD. Statistical analysis for F) was performed using two‐way ANOVA. Other analyses were performed using one‐way ANOVA. p‐values <0.05 was considered significant: ***P <0.001, ns indicated no significance to the positive control (p > 0.05). u.d. indicated undetectable.

Figure 2

Preparation and characterization of CoVR‐MVs. A) Schematics of the constructs of ACE2iRb3 and DPP4iRb3 for generations of ACE2‐ and hDPP4‐overexpressing cell lines. B) Fluorescence confocal images of 293T‐hACE2‐ and 293T‐hDPP4 cells (Scale bar, 25 µm). C) Western blot analyses of expressions of hACE2 and hDPP4 in 293T‐hACE2 and 293T‐hDPP4 cells. D) Schematic illustration for the generation of CoVR‐MVs. E) Representative transmission electron microscopy image (scale bar, left, 1 µm; right, 200 nm), and F) histogram of the particle size distribution of CoVR‐MVs. G) DLS stability assay demonstrated that CoVR‐MVs size remains stable for at least 15 d (n = 3). Data were shown as mean ± SD. H) Western blot analyses of hACE2 and hDPP4 in Mock‐MVs, hACE2‐MVs, hDPP4‐MVs and CoVR‐MVs. I) Fluorescence confocal images of DiD‐hACE2‐MVs and DiO‐hDPP4‐MVs (Scale bar, 10 µm). The fluorescent signal of CoVR‐MVs was detected by flow cytometry. The hACE2‐MVs and hDPP4‐MVs were respectively labeled with DiD and DiO fluorescence dyes before fusion. J) Measurement for the inhibition efficiency of hACE2‐MVs, hDPP4‐MVs, CoVR‐MVs, Mock‐MVs, and soluble hACE2 in a cell‐based SARS‐CoV‐2 spike function blocking test by using fluorescent STG probe and high‐content imaging assay. K) Confocal images of STG, cell membrane and nucleus in 293T‐hACE2 cells at 1 h post STG co‐incubation with each MVs and hACE2, respectively (Scale bar, 20 µm).

Figure 3

CoVR‐MV broadly interferes with the infections of SARS‐CoV, MERS‐CoV, SARS‐CoV‐2 and evolving variants in vitro. A) Neutralizing curves of CoVR‐MVs and control MVs against SARS‐CoV, MERS‐CoV and 18 different SARS‐CoV‐2 variants in LVpp pseudovirus system (n = 3). Y‐axis depicted the percentage of neutralization. Data were shown as mean ± SD. B) List of different viruses and indicated IC₅₀ values. IC₅₀ values quantified by hACE2 concentration. Soluble hACE2 protein was set as the control.

Figure 4

CoVR‐MV therapy promotes endogenous type I IFN production. A) In vivo biodistribution of intranasally administrated DiD‐labeled CoVR‐MVs in the mouse. B) Representative ex vivo fluorescent images and quantitative analysis of major organs from Syrian hamsters at 24 h after intranasal administration with DiD‐labeled CoVR‐MVs (n = 3). C) Confocal images showing the capture of CoVR‐MVs by macrophages in lung tissue of mice (Scale bar, 40 µm). D) CoVR‐MV enhance viral clearance via binding SARS‐CoV‐2 and promote the phagocytosis effect of macrophage. E) Viral RNA levels in macrophages collected at 12 h post coincubation (n = 3). Viral load was measured by RT‐PCR. The primers of SARS‐CoV‐2 ORF1ab and N genes were used. F) Confocal microscopy images of macrophages stained for DAPI (blue), lysosomes (Lyso‐Tracker, green) incubated with CoVR‐MV (DiD, red) for 3 h (Scale bar, 20 µm). D) After the virus‐CoVR‐MV complex was devoured by macrophage, CoVR‐MV restricts SARS‐CoV‐2 replication. H) Relative viral RNA levels in macrophages collected at 1 h and 12 h post co‐incubation, respectively (n = 3). The primers of SARS‐CoV‐2 ORF1ab and N genes were used. Relative mRNA levels of I) DHCR7 and J) IFN‐β in macrophages after co‐incubation of CoVR‐MVs and SARS‐CoV‐2 for 12 h (n = 3). The mRNA levels of DHCR7 and IFN‐β were standardized to the house‐keeping gene γ‐actin. Relative mRNA levels of K) DHCR7 in macrophages and L) concentration of IFN‐β in supernatant after incubation of CoVR‐MVs or MβCD‐MVs for 12 h (n = 3). M) CoVR‐MV mediated upregulation of IRF3 and IFN‐β by inhibiting DHCR7. Statistical analysis for (E,I,J) were performed using one‐way ANOVA. Statistical analysis for (H,K,L) were performed using unpaired t‐test. p‐values <0.05 was considered significant: *P < 0.05, **P <0.01, ***P <0.001, ns indicated no significance to the positive control (p > 0.05). u.d. indicated undetectable.

Figure 5

CoVR‐MV reverses the imbalanced innate immune responses in SARS‐CoV‐2 infected Syrian hamsters. Relative mRNA levels of A) IFN‐α, B) IFN‐β and C) MX1, and proinflammatory cytokines include D) IL‐6, E) TNF‐α and (F) IFN‐γ in the lung tissues collected from euthanized hamsters at 1, 3, and 5 dpi, respectively (n = 5). The mRNA levels were standardized to the house‐keeping gene γ‐actin. G) Schematic illustration for CoVR‐MVs mediated rebalance of the dysregulated innate immune response. H) Body weight changes of the survived hamsters from 0 to 5 dpi were recorded (n = 5). Representative I) gross images and J) H&E staining images for lung lobes collected at 5 dpi were shown. Scale bar = 2 mm. H&E staining images for all of the remaining hamster lobes were shown in Figure S13 (Supporting Information). K) Comprehensive pathological scores for lung sections were determined based on the severity and percentage of injured areas for each lung lobe (details shown in Table S1, Supporting Information). L) Viral RNA levels in turbinate, trachea and lung tissues collected at 5 dpi were measured by RT‐PCR (n = 5). The primers of SARS‐CoV‐2 ORF1ab gene were used. Data were shown as mean ± SD. Statistical analysis for H) was performed using two‐way ANOVA. All other analyses were conducted using one‐way ANOVA. p‐values <0.05 was considered significant: *P < 0.05, **P < 0.01, ***P < 0.001, ns indicated no significance to the positive control (p > 0.05). u.d. indicated undetectable.

Figure 6

CoVR‐MV protects hamsters from lethal ARDS of SARS‐CoV‐2 variant infections. A) Schematic diagram of SARS‐CoV‐2 infection and CoVR‐MVs therapy. Hamsters were intranasally inoculated with 1 × 10⁴ PFU of prototype SARS‐CoV‐2, 614G, and B.1.351 variants, respectively. The infected hamsters treated with saline were set as controls. B) Survival analysis (n = 10). C) Body weight changes of the survived hamsters from 0 to 5 dpi were recorded (n = 5). Representative D) gross images and E) H&E staining images for lung lobes collected at 5 dpi were shown. Scale bar = 2 mm. H&E staining images for all of the remaining hamster lobes were shown in Figure S15 (Supporting Information). F) Comprehensive pathological scores for lung sections were determined based on the severity and percentage of injured areas for each lung lobe (details shown in Table S2, Supporting Information). Viral RNA levels in G) turbinate, H) trachea and I) lung tissues collected at 5 dpi were measured by RT‐PCR (n = 5). The primers of SARS‐CoV‐2 ORF1ab gene were used. Data were shown as mean ± SD. Statistical analysis for C) was performed using two‐way ANOVA. All other analyses were conducted using one‐way ANOVA. p‐values <0.05 was considered significant: *P < 0.05, **P < 0.01, ***P < 0.001, ns indicated no significance to the positive control (p> 0.05). u.d. indicated undetectable.