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. 2022 Feb 22;119(8):e2111600119.
doi: 10.1073/pnas.2111600119.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection

Jonas Schuhenn  1 , Toni Luise Meister  2 , Daniel Todt  2   3 , Thilo Bracht  4   5 , Karin Schork  4   6 , Jean-Noel Billaud  7 , Carina Elsner  1 , Natalie Heinen  2 , Zehra Karakoese  1 , Sibylle Haid  8 , Sriram Kumar  9 , Linda Brunotte  9   10 , Martin Eisenacher  4   6 , Yunyun Di  11 , Jocelyne Lew  12 , Darryl Falzarano  12 , Jieliang Chen  13 , Zhenghong Yuan  13 , Thomas Pietschmann  8   14   15 , Bettina Wiegmann  16 , Hendrik Uebner  17 , Christian Taube  17 , Vu Thuy Khanh Le-Trilling  1 , Mirko Trilling  1 , Adalbert Krawczyk  1   18 , Stephan Ludwig  9   10 , Barbara Sitek  4   5 , Eike Steinmann  2 , Ulf Dittmer  1 , Kerry J Lavender  19 , Kathrin Sutter  20 , Stephanie Pfaender  21
Affiliations

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection

Jonas Schuhenn et al. Proc Natl Acad Sci U S A. .

Abstract

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Keywords: SARS-CoV-2; antiviral; immunotherapy; type I IFNs.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Treatment with IFNα subtypes reveals distinct antiviral effects against SARS-CoV-2. (A) Antiviral activity of IFNα subtypes (100 U/mL or 1,000 U/mL) and IFNλ3 (100 ng/mL or 1,000 ng/mL) against SARS-CoV-2 on VeroE6 cells (TCID50 per milliliter). (B) Representative immunofluorescence staining of IFN-treated SARS-CoV-2–infected VeroE6 cells. (Scale bar: 100 µm.) (C–F) IFNα subtypes were titrated against SARS-CoV-2 on VeroE6 cells by TCID50 assay, and the IFNs were grouped in high (C), medium (D), and low (E) antiviral patterns, and the mean values of each group are plotted in F. (G and H) Antiviral activity of IFNα subtypes and IFNλ3 in SARS-CoV-2–infected primary hAECs at 72 h p.i. (G) and kinetics of four selected IFNs (H). (I and J) Antiviral activity of IFNα subtypes and IFNλ3 in Influenza A/PR8-infected primary hAECs at different time points p.i. Mean values of high (I) and low/not (J) antiviral IFNs are shown. In A and G, each data point represents a biological replicate or an individual donor. In A, C–F, I, and J, mean values ± SEM are shown for n = 3. In G and H, n = 4 to 7. Statistical tests were performed for the individual IFN-treated groups against the untreated control group. *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig. 2.
Fig. 2.
Transcriptomic analyses display IFN subtype–specific immune signatures. Transcriptomic analyses of IFN-treated (16 h posttreatment; 1,000 U/mL or 1,000 ng/mL) or SARS-CoV-2–infected (18 h p.i.) hAECs. (A) Biological processes induced by IFNs or SARS-CoV-2. (B) Heat maps displaying genes contained in antiviral response. (C) UpSet plots to summarize key DEGs. Numbers of individual or group-specific DEGs are shown as bars and numbers. The bottom right horizontal bar graph labeled Set Size shows the total number of DEGs per treatment. IFNs are plotted, according to their antiviral activity, in three groups (high, medium, and low). (D) Heatmap of the 19 basal DEGs expressed by all IFNs as identified in C. (E) Plot depicting fold changes (FC) of the identified 42 unique genes in the group displaying high antiviral activity and association of genes to functional categories. In A–E, n = 4.
Fig. 3.
Fig. 3.
Proteomic analysis highlights key cellular mediators. Proteomic analysis of IFN-treated (1,000 U/mL or 1,000 ng/mL) and/or SARS-CoV-2–infected hAECs. (A) Biological processes induced by IFNs 16 h posttreatment (t = 0 h) or 88 h posttreatment (t = 72 h). (B) Volcano plots of IFN-treated hAECs at different time points posttreatment. Detected ISGs are colored yellow. (C) Biological processes induced by IFNs 88 h posttreatment in the presence of SARS-CoV-2 (t = 72 h); mRNA, messenger RNA; ncRNA, noncoding RNA. (D) Volcano plots of IFN-treated SARS-CoV-2–infected hAEC. Detected proteins are colored due to their biological function: red, complement activation; green, O-glycan processing. (E) Heatmaps of differentially activated biological processes by highly antiviral IFNα5 and IFNλ3 compared to untreated controls at different time points posttreatment in the presence and absence of SARS-CoV-2. (F) STRING analysis of proteins increased in IFN-treated and/or SARS-CoV-2–infected hAECs and identified abundant protein–protein interactions. Proteins are shown as circles and colors indicating biological processes. In A–F, n = 4.
Fig. 4.
Fig. 4.
Therapeutic potential of highly antiviral IFNα subtypes. (A) Schematic depiction of treatment. (B) Pretreatments and posttreatments with IFNα5 of VeroE6 cells by icELISA (gray bars) and TCID50 assay (white bars). Each data point represents a biological replicate measured at an optical density at 450 nm (OD450). (C) Inhibition of SARS-CoV-2 infection by IFNα5 and analysis of drug combination experiments using SynergyFinder web application (72) 16 h before infection (Pre-Treatment). (D) Inhibition of SARS-CoV-2 infection and analysis of drug combination experiments using SynergyFinder web application 8 h p.i. (Post-Treatment). (E) Inhibition of SARS-CoV-2 infection by clinically approved IFNα2 and analysis of drug combination experiments using SynergyFinder web application 16 h before infection (Pre-Treatment). (F) Inhibition of SARS-CoV-2 infection and analysis of drug combination experiments using SynergyFinder web application 8 h p.i. (Post-Treatment). (G–I) Remdesivir and IFNα5 combinational treatment 8 h p.i. of hAECs with low doses (0.313 μM remdesivir, 0.2444 U/mL IFNα5; G), medium doses (0.63 μM remdesivir, 15.625 U/mL IFNα5; H) and high doses (2.5 μM remdesivir, 1.953 U/mL IFNα5; I). (J) Therapeutic effect of IFNα5 and IFNα2 in SARS-CoV-2–infected LoM. In B–I, n = 3. In J, n =9. *P < 0.05; **P < 0.01.
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