Interferon generated by airway replication circulates, primes distal organs and is what keeps SARS-CoV-2 respiratory
lab-led
Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity
lab-ledDiscovery
Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.
- Senior authors
- Benjamin R. tenOever
- Correspondence
- Benjamin R. tenOever
Research areas & themes
Innate Immune Signaling and the Interferon ResponsePandemic Host Response and DiseaseCalibration of the Interferon Response In VivoInterferon as Intervention and Systemic PrimingModels for Pandemic Virology
Citation
Hoagland DA, Møller R, Uhl SA, Oishi K, Frere J, Golynker I, Horiuchi S, Panis M, Blanco-Melo D, Sachs D, Arkun K, Lim JK, tenOever BR. Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity. Immunity. 2021. Volume 54, issue 3, pages 557-570.e5.
DOI 10.1016/j.immuni.2021.01.017. PMID 33577760. PMCID PMC7846242.
One-sentence contribution
Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.
Executive summary
Severe COVID-19 is associated with a host response in which type I interferon induction is muted while chemokine expression is high, and this combination has been proposed to drive the neutrophil and monocyte infiltration that characterizes the disease. Testing that idea and any intervention derived from it requires an animal model in which an unmodified clinical isolate causes progressive lower respiratory tract disease. The authors used golden hamsters, which are naturally permissive, and profiled infection across time and across tissues by mRNA sequencing, quantitative RT-PCR, plaque assay and histopathology. Comparison against pandemic H1N1 influenza A virus at matched viral loads showed that SARS-CoV-2 drives a stronger inflammatory and neutrophil associated signature. Infection could be established by intranasal, ocular, contact and, less efficiently, fomite exposure, and as few as ten plaque forming units sufficed to seed the lower respiratory tract. A longitudinal series showed interferon stimulated gene and chemokine induction that peaked in the trachea several days before the lungs, and an antiviral transcriptional response in olfactory bulb, brain and small intestine despite viral reads orders of magnitude below respiratory levels. Intranasal universal interferon alpha A/D, given prophylactically or starting one day after infection, reduced infectious virus, reduced proinflammatory transcripts, shifted the lung infiltrate away from neutrophils, and prevented transmission in three of five contact exposed animals. An intranasal double-stranded RNA mimetic gave comparable antiviral activity.
Scientific context
By late 2020 several groups had reported that SARS-CoV-2 elicits an unbalanced host response in which interferon induction is low relative to chemokine induction, and this had been described in cell culture, in a ferret model, in autopsy material and in patient cohorts. Severe COVID-19 had also been linked to autoantibodies against interferons and to inborn errors of innate immunity affecting TLR3 and IRF7, which together argued that an inadequate interferon response contributes to disease severity. At the same time systemic interferon administration had shown poor concordance in trials and can produce unwanted effects, and the SOLIDARITY trial did not show a mortality benefit. What remained undefined, as the paper states, was the transcriptional host response across the full course of infection and across tissues, including early infection and the period after viral clearance, because clinical sampling cannot readily provide it. Golden hamsters had been introduced as a permissive small animal model, but the hamster genome annotation was incomplete for immune genes, including Ifnb1, which limited transcriptomic interpretation.
Central question
What is the systemic and longitudinal host response to SARS-CoV-2 across respiratory and distal tissues, and can locally delivered type I interferon, rather than systemic interferon, be used to limit viral replication, tissue damage and transmission?
Experimental strategy
The design pairs a descriptive arm with an interventional arm in the same model. Golden hamsters were chosen because an unmodified clinical isolate replicates and causes progressive lower respiratory tract pathology without host genetic modification, which avoids the confounds of transgenic or adenovirus transduced ACE2 mouse systems. A matched influenza A virus comparison at equivalent viral read depth was used to separate what is specific to SARS-CoV-2 biology from what accompanies any respiratory virus at that load. Route and dose were varied deliberately, since the amount and site of the inoculum shape the response and the authors wanted to know which route is reliable before committing to it for the rest of the study. A longitudinal bulk mRNA sequencing series across trachea, lung, olfactory bulb, brain and small intestine at two inoculum doses provides the temporal and spatial map, with curated interferon stimulated gene and chemokine lists and gene ontology enrichment as the readouts. Because Ifnb1 is not annotated in the golden hamster genome, the authors performed a de novo assembly, identified a candidate transcript, cloned it, and showed it induces interferon stimulated genes in hamster but not human cells, which makes interferon beta measurable in this model. Sensitive subgenomic RNA assays combined with plaque assay were used to distinguish replicating virus from viral RNA in distal tissues. For the intervention, a universal interferon alpha A/D preparation was selected after comparing candidate interferons for potency on hamster cells, then delivered intranasally in prophylactic, therapeutic and contact transmission designs.
Key findings
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At comparable viral loads five days after infection, SARS-CoV-2 and influenza A virus produced distinct lung transcriptional signatures, with SARS-CoV-2 driving higher inflammatory transcripts including Ccl5, Gzmb and Il1rn and higher neutrophil associated transcripts Fcgr3, Ccrl2 and Cf2 (Figures 1A to 1C).
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A functional golden hamster Ifnb1 transcript was identified by de novo assembly and validated by cloning and supernatant transfer, which induced interferon stimulated genes in hamster but not human cells (Figures S1A to S1E). Using this annotation, no significant Ifnb1 induction was detected in either SARS-CoV-2 or influenza A virus infected animals (Figure S1F), which the authors interpret as high chemokine expression in the setting of a muted type I interferon response.
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Infection was established by intranasal inoculation, ocular inoculation and direct contact, with fomite exposure the least efficient route, and all routes including the ocular route produced a respiratory host response with elevated Isg15 and Cxcl11 (Figures 2B, 2C and S2A).
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Ten plaque forming units were sufficient to establish lower respiratory tract infection. Raising the inoculum as high as one hundred thousand plaque forming units did not increase replication and tended to lower subgenomic nucleocapsid and nsp14 levels, while eliciting a comparable host response (Figures 2D, 2E, S2B and S2C). Intranasal infection was reproducible across twelve animals (Figures 2F to 2H).
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Infection produced progressive lower respiratory tract pathology scored by a certified pathologist, with nucleocapsid protein maximal at day four and still detectable at day fourteen, epithelial degeneration and alveolar necrosis, type II pneumocyte hyperplasia peaking at days four and eight, cumulative inflammation maximal at day eight, and atypical adenomatous hyperplasia at day fourteen after most inflammation had resolved (Figures 3A to 3E). Apoptosis in bronchial epithelium, nested neutrophil accumulations and severe vascular edema were observed at day three (Figures 3F to 3H).
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The interferon stimulated gene response tracked viral load in the trachea but did not contain replication, and high lung viral load followed on day four (Figures 4A and 4B). A hundredfold higher inoculum removed the delay in interferon stimulated gene induction in both tissues without restricting the virus. Irrespective of dose, induction of many classical interferon stimulated genes appeared limited.
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Inflammation moved from the upper to the lower respiratory tract. With a one hundred plaque forming unit challenge, Ccl4 and Ccl5 peaked at day two in trachea but not until day six in lung, and the tracheal peak preceded the lung peak by roughly four days (Figures 5A, 5B and S5A). Tnf and Il1b were high in the upper tract and largely absent in the lower, while Il36a, Cxcl6 and Ccl22 were higher in lung. Detectable Ifnb and Ifnl reads appeared only in trachea one day after a high dose infection and in no other tissue at any time (Figures 5C and 5D). Cytokine expression fell by day fourteen, coinciding with peak spike specific serum IgG (Figure 5E).
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Distal tissues showed a strong antiviral transcriptional response despite viral reads orders of magnitude below the respiratory tract. Interferon signaling enrichment was evident in brain, olfactory bulb and small intestine, brain differential expression peaked at day two coinciding with the upper respiratory tract viral peak, and the most sustained interferon response was in the olfactory bulb (Figures 6A to 6F and S6).
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Subgenomic nucleocapsid RNA was amplifiable in olfactory bulb, brain and small intestine on days one, two and four, and as far out as day eight in olfactory bulb at roughly one two hundredth of day four lung levels, but did not consistently correlate with infectious particles by plaque assay (Figures 6G and 6H). The authors interpret this as suggesting that dissemination of virus derived pathogen associated molecular patterns, rather than productive infection, may underlie the distal inflammation, and they state that further study is needed to substantiate it.
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Prophylactic intranasal interferon alpha A/D induced a broad interferon stimulated gene program in hamster lung (Figure 7A, Table S4) and, when started twenty four hours before challenge, reduced infectious virus and subgenomic nucleocapsid RNA and lowered Cxcl11 and Il6 (Figures 7B to 7E), with reduced nucleocapsid and increased MxA protein by immunohistochemistry and less inflammation by histology (Figures 7F to 7H).
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Therapeutic intranasal interferon alpha A/D started twenty four hours after infection reduced infectious virions and raised Il10 at day three, and at day six reduced subgenomic membrane and nucleocapsid RNA and Il6, with an infiltrate weighted toward reactive macrophages rather than neutrophils and increased bronchial epithelial mitosis (Figures 7I to 7M).
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In a contact transmission design, prophylactic intranasal interferon alpha A/D reduced titers in infected animals by more than two logs and prevented transmission in three of five exposed animals, corroborated by nucleocapsid and MxA immunohistochemistry and by lung subgenomic nucleocapsid levels (Figures S7E to S7H). Intranasal administration of a double-stranded RNA mimetic gave antiviral activity comparable to interferon alpha A/D (Figure S7I).
Mechanistic model
The study does not establish a definitive mechanism for either the systemic inflammation or the protective effect of local interferon. What the data support is a sequence rather than a molecular chain. Virus replicates first and most heavily in the upper respiratory tract, where chemokine and interferon stimulated gene induction begins, then spreads to the lower respiratory tract where the same programs appear several days later, and inflammation appears in olfactory bulb, brain and small intestine at times that track the upper respiratory tract viral peak rather than local replication. For the distal inflammation the authors propose, explicitly as speculation, that abundant subgenomic viral RNA, possibly protected as a nucleocapsid bound ribonucleoprotein, is secreted or spills over from the primary replication site and acts as a disseminated pathogen associated molecular pattern. The supporting observation is the discordance between subgenomic nucleocapsid detection and infectious particle recovery, which is suggestive rather than demonstrative, since neither transfer of such material nor its recognition by a specific sensor was tested. For the intervention, the data show that raising the interferon stimulated gene set in the airway before or shortly after challenge lowers viral load, lowers proinflammatory transcripts, and changes the composition of the infiltrate. Whether the reduced neutrophil presence follows from the lower viral load, from a direct effect of interferon on recruitment, or from both is not resolved. The authors also note it remains unclear whether the heightened distal antiviral state itself restricts viral tropism in this model.
Conceptual or technical advance
The study provides a longitudinal, multi tissue transcriptional atlas of SARS-CoV-2 infection in a naturally permissive small animal, annotated well enough to read interferon biology, since the previously unannotated hamster Ifnb1 was identified and functionally validated as part of the work. That makes the golden hamster usable for interferon focused questions rather than only for viral load and pathology endpoints. Conceptually, the separation between where virus replicates and where inflammation appears reframes distal COVID-19 manifestations as potentially driven by disseminated viral material rather than requiring distal productive infection. Practically, the work moves interferon from a systemic therapeutic, where tolerability has limited it, to a local airway prophylactic and early therapeutic, and shows that the same benefit can be obtained by delivering a pattern recognition receptor agonist that induces endogenous interferon, which the authors note is cheaper and easier to manufacture and store.
Relationship to the broader research program
The authors state their general strategy as developing broad-spectrum antiviral countermeasures that exploit the host response rather than pathogen specific interventions for each new outbreak, and they frame airway delivery of type I interferon as an instance of that strategy. The work connects directly to the laboratory's earlier description of an imbalanced host response to SARS-CoV-2 across cell, ferret and patient systems, which it extends into a systemic and longitudinal frame. The proposal that viral RNA can act as a disseminated pathogen associated molecular pattern is linked by the authors to their own earlier work on unrelated RNA viruses. Category 3 synthesis, requiring several corpus papers side by side, is that the laboratory repeatedly builds animal models in which the host transcriptional response, rather than viral titer alone, is the primary readout, and then intervenes on that response. That statement is not asserted from this paper alone.
Related publications
- Blanco-Melo et al. 2020, imbalanced host response to SARS-CoV-2 drives development of COVID-19, predecessor. Cited throughout as the source of the low interferon and high chemokine framing and of the ferret model comparison, and shares authors with this study.
- Langlois et al. 2013, microRNA based strategy to mitigate the risk of gain of function influenza studies, methodological foundation. Appears in the reference list as prior laboratory work on engineered antiviral approaches.
- tenOever et al. 2002 and 2004, cited by the authors as prior support for the idea that viral RNA species can be disseminated and detected apart from infectious particles, conceptual extension.
- Si et al. 2020, human organ chip enabled pipeline to rapidly repurpose therapeutics during viral pandemics, companion. Shares several authors and belongs to the same effort to identify host directed countermeasures.
Limitations and boundaries
The authors provide their own limitations section. The study uses young golden hamsters that clear the virus and survive, so it does not represent older animals or lethal disease, and both age and severity may alter the host response and tropism. Extrapolation to human disease is explicitly flagged. The value of intranasal interferon late in the disease course was not tested, and the authors call for work in a lethal model and in animals genetically lacking innate immune sensors such as TLR3 and TLR7. Beyond the stated limitations, the transcriptomics are bulk rather than single cell, so cell type attributions such as T cell or macrophage origin of particular transcripts are inferences from marker genes rather than direct measurements. Distal tissue conclusions rest on small group sizes, with as few as two animals in one small intestine condition, and on pooled RNA for some subgenomic RNA measurements. The evidence that disseminated viral RNA drives distal inflammation is circumstantial. Hamster genome annotation is incomplete, gene names are given by human ortholog, and the Ifnb1 transcript used here was newly assigned by the authors. The inoculum dose strongly shapes the response, which the authors note should be considered before extrapolating any of these data to human disease. Finally, the interferon intervention was tested as intranasal administration in a rodent airway and does not establish efficacy or dosing in humans.
Audience summaries
25 words
Hamsters infected with SARS-CoV-2 develop inflammation in organs the virus barely reaches, and interferon sprayed into the nose before or after exposure lowers virus and disease.
75 words
SARS-CoV-2 provokes strong chemokine signaling but weak interferon signaling, a combination linked to severe COVID-19. Profiling infected golden hamsters across tissues and time showed inflammation moving from the upper to the lower airway and appearing in brain, olfactory bulb and intestine despite almost no virus there. Delivering type I interferon directly into the nose, before or one day after infection, reduced viral load, tissue damage and onward transmission.
150 words
Golden hamsters infected with an unmodified clinical SARS-CoV-2 isolate were profiled longitudinally by mRNA sequencing, quantitative RT-PCR, plaque assay and histopathology across trachea, lung, olfactory bulb, brain and small intestine. Compared against influenza A virus at matched viral load, SARS-CoV-2 drove a stronger inflammatory and neutrophil associated signature. A newly assembled and functionally validated hamster interferon beta gene allowed interferon induction to be measured, and it remained low while chemokines rose. Interferon stimulated gene and chemokine peaks occurred in the trachea several days before the lung, and antiviral programs appeared in distal organs where infectious virus was scarce, which the authors attribute tentatively to disseminated viral RNA acting as a pattern recognition ligand. Intranasal universal interferon alpha A/D given before challenge, or beginning one day after, lowered infectious virus and inflammatory transcripts, shifted the lung infiltrate toward macrophages, and blocked transmission in three of five contact exposed animals.
Discoveries supported by this paper
Documented publication relationships
- Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells — companion.
- SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium — predecessor.
- Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19 — predecessor.
- SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery — methodological foundation.
- A diminished immune response underlies age-related SARS-CoV-2 pathologies — predecessor.
- Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia — methodological foundation.
- Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia — predecessor.
- Delayed engagement of host defenses enables SARS-CoV-2 viremia and productive infection of distal organs in the hamster model of COVID-19 — predecessor.
- Mouse genome rewriting and tailoring of three important disease loci — predecessor.