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. 2021 Aug 9;17(8):e1009427. doi: 10.1371/journal.ppat.1009427

Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model

Pierre Bessière 1, Marine Wasniewski 2,#, Evelyne Picard-Meyer 2,#, Alexandre Servat 2,#, Thomas Figueroa 1, Charlotte Foret-Lucas 1, Amelia Coggon 1, Sandrine Lesellier 3, Frank Boué 2, Nathan Cebron 1, Blandine Gausserès 1, Catherine Trumel 4, Gilles Foucras 1, Francisco J Salguero 5, Elodie Monchatre-Leroy 2, Romain Volmer 1,*
Editor: Michael S Diamond6
PMCID: PMC8376007  PMID: 34370799

Abstract

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.

Author summary

Type I interferons are major antiviral effectors produced by the host in response to viral infections. Importantly, delayed or impaired type I IFN signalling response has been shown to correlate with severe COVID-19. These observations provided further impetus to test the administration of exogenous type I IFN as a treatment against SARS-CoV-2 infection in patients. However, studies using MERS-CoV or SARS-CoV infected mice demonstrated that type I interferon treatment was beneficial when administered early, but was ineffective and even caused deleterious immunopathology when administered at later stages of infection. It is therefore crucial to understand how the timing of the type I IFN treatments modulates their efficacy and safety against SARS-CoV-2. In this preclinical study using the SARS-CoV-2-infected Syrian hamster model, we showed that intranasal type I IFN treatment was beneficial only when administered before the onset of symptoms. Importantly, late treatment was ineffective but was not associated with deleterious effects. This study provides important information to interpret clinical trials showing no to modest effects of type I IFNs in COVID-19 patients.

Introduction

Type I interferons (IFNs) are major antiviral cytokines and their finely tuned production is critical for host protection against viruses [1]. In vitro studies demonstrated that SARS-CoV-2 was very sensitive to the antiviral effects of type I IFN [2–4]. In addition, development of severe COVID-19 was shown to correlate with decreased type I IFNs production or impaired type I IFN signaling [5–9]. In agreement with these observations, recombinant type I IFNs are being tested in a number of clinical trials to treat COVID-19 patients [10–12]. However, the design and interpretation of these clinical trials need to consider that the timing of type I IFN treatment may be critical for its efficacy and safety against SARS-CoV-2 [13,14]. Indeed, studies in SARS-CoV and MERS-CoV infected mice demonstrated that type I IFN-treatment was beneficial when administered early, while it was deleterious when administered at later stages of infection [15,16]. How the timing of type I IFN treatment modulates clinical efficacy against SARS-CoV-2 is currently unknown and needs to be tested in an animal model.

Results

To address this question, we first determined the consequences of recombinant universal IFN-α (IFN) (Hu-IFN-αA/D[Bg/II], pbl assay science, Piscataway, NJ) on the expression of the type I IFN stimulated gene (ISG) Mx1. Mx1 is an ISG and its level of expression is a good indicator of the levels of type I and type III IFN acting locally [17]. We observed a significant upregulation of Mx1 expression in the nasal turbinates, lungs and spleen of hamsters treated intranasally with 105 IU IFN, demonstrating that this molecule was active in hamsters (Fig 1A). Pulmonary Mx1 gene expression 24 hours post IFN treatment did not differ significantly between animals treated with 105 IU IFN or with 7.105 IU IFN (Fig 1B). At 48 hours post treatment with 105 IU IFN, pulmonary Mx1 mRNA expression was reduced compared to 24 hours post treatment with the same dose, but remained upregulated compared to placebo treatment (Fig 1B). Next, we analyzed Mx1 protein expression in the lungs of IFN-treated hamsters by immunohistochemistry. In IFN-treated hamsters, Mx1 protein expression was detected in the main target cells of SARS-CoV-2, including pneumocytes, bronchiolar and bronchial epithelial cells, but also in endothelial cells and immune cells within the lung parenchyma (Fig 1C). The percentage of Mx1 positive lungs was significantly increased 24 hours post-treatment in animals administered 105 IU IFN and further increased in animals administered 7.105 IU IFN (Fig 1D). Importantly, in animals administered 105 IU IFN, Mx1 positive lung area was equivalent at 24 hours and 48 hours post-treatment, indicating that Mx1 protein levels remained elevated for 48 hours following type I IFN intranasal administration (Fig 1D), in accordance with a previous report [18]. We thus decided to treat hamsters every two days in an effort to minimize the side effects due to the anesthesia required to treat hamsters intranasally with IFN. In human clinical trials, nebulized type I IFNs are being tested at 6.106 IU per treatment, which corresponds to a hamster equivalent dose of approximately 105 IU per hamster based on body surface area conversion, as described in Materials and Methods [12,19]. We therefore treated hamsters with 105 IU IFN per hamster in the following experiments.

Fig 1. Impact of IFN-α treatment on Mx1 lung transcript and protein levels in non-infected hamsters.

Fig 1

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(A) Syrian hamsters were treated intranasally either with placebo or with 105 IU recombinant universal IFN-α (IFN). Tissues were harvested at day 1 post-treatment. Transcripts levels of Mx1 relative to the housekeeping genes RPL18 and RPS6KB1 were determined by RT-qPCR. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test. (B-D) Syrian hamsters were treated intranasally either with placebo or with 105 UI IFN or with 7.105 IU IFN. Tissues were harvested either at day 1 or day 2 post-treatment. (B) Lung transcripts levels of Mx1 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. (C) Representative pictures were selected to display Mx1 lung protein levels detected by immuno-chemistry (IHC). Scale bar: 100μm. (D) Quantification of percent lung area positive for Mx1 protein detected by IHC. D0: day 0; D1: day 1; D2; day 2. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

Next, we designed a study that evaluated the prophylactic and therapeutic efficacy of intranasally administered recombinant IFN against SARS-CoV-2 infection in Syrian hamsters (Fig 2A). Hamsters intranasally infected with a high SARS-CoV-2 dose develop clinical disease caused by lung pathology, which closely mirrors severe human COVID-19 [20,21]. Following challenge with 104 TCID50 SARS-CoV-2, we observed significant weight loss in the placebo-treated animals, as expected with a high SARS-CoV-2 inoculum titer [20,21]. No protection from weight loss was observed in the IFN-late group, for which treatment was initiated at the onset of clinical signs, when infected animals started to significantly lose weight three days post-infection (Fig 2B). By contrast, we observed a significant protection from weight loss in the IFN-pre group (prophylactic treatment initiated 16 hours before infection) and in the IFN-early group (treatment initiated at one day post-infection) compared to the placebo group (Fig 2B). The protection from weight loss in the IFN-pre and in the IFN-early groups was not associated with a reduction of viral excretion level or duration, as viral RNA levels measured by RT-qPCR from oropharyngeal swabs were similar in all groups (Fig 2C). In agreement with this observation, subgenomic viral RNA levels in the nasal turbinates were similar in all groups (S1 Fig). As SARS-CoV-2 respiratory disease is due to lower respiratory tract damage, we analyzed viral load in the lungs. We detected a reduction of pulmonary viral subgenomic RNA levels and infectious viral titers in all the IFN-treated groups at day 5 post-infection, compared to the placebo group, which reached statistical significance in the IFN-early group only (Fig 2D and 2E).

Fig 2. Impact of type I IFN-α treatment on SARS-CoV-2-induced weight loss and viral titers.

Fig 2

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(A) Overview study design. (B) Percentage of body weight change with weight measured day 1 pre-infection before the IFN-pre treatment set as the reference weight (6 animals per group). Statistical analysis: two-way ANOVA comparing treatment effects with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test. (C). Viral genomic RNA in oropharyngeal swabs (6 animals per group). The dotted line indicates limit of detection. (D and E) Lungs viral titers determined by RT-qPCR targeting viral sgRNA relative to the housekeeping genes RPL18 and RPS6KB1 (D) and by TCID50 (E). D2: day 2 post infection; D5: day 5 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

Evaluation of the respiratory tract from infected animals revealed a mild to moderate bronchointerstitial pneumonia at day 2 post-infection, progressing to moderate/severe with lung consolidation at day 5 and resolving at day 15 with only small lesioned areas remaining, as previously observed [21]. The lesions were characterized by infiltrates of macrophages and neutrophils, with fewer lymphocytes and plasma cells (Figs 3A and S2). A reduction of the lung pathology scores was observed in the IFN-treated groups compared to the placebo group, which reached statistical significance in the IFN-early group only (Fig 3B). RNAScope in situ hybridization (ISH) was used to determine the localization of viral RNA in the lungs of infected animals. Viral RNA was observed in bronchial and bronchiolar epithelial cells and in regions of inflammatory infiltrates at day 2 post-infection (S2 Fig). The viral RNA positive area diminished at day 5 and coincided with inflammatory infiltrates. Quantification of viral RNA positive area revealed a slight non-statistically significant reduction of viral RNA in the IFN-pre and in the IFN-early groups at day 2 and 5 post-infection compared to the placebo group (Fig 3C). Mx1 protein was upregulated in the lungs of infected hamsters, as detected by immunohistochemistry, and the percentage of Mx1 positive lung was equivalent in placebo and IFN-treated hamsters (Figs 3D and S2). Finally, hematological analyses revealed a modest lymphocytopenia in SARS-CoV-2 infected hamsters, with no difference between the IFN-treated groups and the placebo group (S3 Fig).

Fig 3. Histopathological analysis of the impact of type I IFN-α treatment.

Fig 3

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(A) Representative pictures were selected to display the pathology from haematoxylin and eosin (H&E) stained lung section from animals at day 2 post infection. Scale bar: 100μm. (B) Severity of lung pathology based on lesional scores evaluated from haematoxylin and eosin (H&E) stained lung section. Statistical analysis: Mann-Whitney test. (C) Quantification of percent lung area positive for viral RNA in lung sections stained with RNAScope in situ hybridization (ISH). Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test. (D) Quantification of percent lung area positive for Mx1 protein detected by immunohistochemistry (IHC). Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM.

To explore the consequences of type I IFN administration on the immune response to SARS-CoV-2 infection, we analyzed the gene expression of immune markers gene expression from the lungs of animals euthanized at day 2 and 5 post-infection. Compared to the non-infected animals, all the infected groups presented a significant upregulation of the ISGs Mx1 and ISG15 and of the C–X–C motif chemokine ligand 10 (CXCL10) messenger RNA (mRNA) expression at day 2 and 5 post-infection, with no difference between the placebo and the IFN-treated groups (Fig 4A). The mRNA expression levels of IFN-γ, and the interleukins (ILs) IL-10 and IL-6 were also significantly upregulated in the infected animals at day 5 post-infection. Similar results were obtained for other immune markers analyzed by RT-qPCR in the lungs (S4 Fig), nasal turbinates (S5 Fig) and spleen (S6 Fig). We also measured the protein levels of chemokine and cytokines either in the lungs or plasma using a commercial enzyme-linked immunosorbent assay (ELISA) directed against hamster IL-6 or a custom-developed hamster multiplex assay. Compared to non-infected animals, we detected an upregulation of CXCL10 and IL-10 protein levels in the lung of all infected groups, with no difference between the placebo and the IFN-treated groups (Fig 4B). We detected a reduction of lung IL-1β levels in IFN-treated groups compared to placebo. Interestingly, lung IL-6 protein level and plasmatic chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNF-α) protein levels were upregulated in the IFN-late group, compared to the IFN-pre and IFN-early groups (Fig 4B and 4C).

Fig 4. Impact of type I IFN-α treatment on the immune response to SARS-CoV-2.

Fig 4

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(A) Lung transcripts levels of Mx1, ISG15, IFN-γ, CXCL10, IL-6 and IL-10 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. (B) Lung protein levels for CXCL10, IL-6, IL-10 and IL-1β protein levels determined by ELISA or a multiplex assay. (C) Plasmatic protein levels for CCL2 and TNF-α determined by a multiplex assay. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

Discussion

In this study, we assessed the in vivo prophylactic and therapeutic efficacy of type I IFN treatment against SARS-CoV-2 infection in the hamster model. Our study demonstrates that type I IFN treatment is beneficial when administered prophylactically or one day post-infection. We observed a significant protection from weight loss in the IFN-pre and in the IFN-early groups, which was associated with a modest reduction of lung viral titers. Interestingly, prophylactic intranasal administration of type I IFN has recently been shown to significantly reduce SARS-CoV-2 replication in hamsters [22]. However, this study did not provide evidence of a protection from weight loss, likely because the inoculum dose of 102 plaque forming units SARS-CoV-2 was not associated with overt clinical signs [22]. We chose a high SARS-CoV-2 inoculum dose of 104 TCID50 to induce clinical signs and significant weight loss, in an effort to model patients requiring therapy. The modest reduction in lung viral titers observed upon prophylactic type I IFN treatment in our study is unlikely due to the dose of type I IFN, 105 IU in our study, versus 2.105 IU in [22], nor to the treatment frequency as we observed sustained Mx1 protein expression over 48 hours in the non-infected IFN treated hamsters (Fig 1D). By contrast, we hypothesize that the modest reduction in lung viral titers observed upon prophylactic type I IFN treatment in our study could be due to the fact that we used a high viral inoculum. Interestingly, a similar observation was made in MERS-CoV infected mice: a significant reduction of viral titers was observed upon early treatment with interferon-β in mice inoculated with 750 pfu MERS-CoV [16], while no reduction was observed in mice treated prophylactically with interferon-β and infected with 5.104 pfu MERS-CoV [23]. Intranasal treatment with type I IFN at day one post-infection reduced clinical signs as efficiently as prophylactic treatment in SARS-CoV-2 infected hamsters. Similar findings were obtained when SARS-CoV-2-infected mice were treated prophylactically or at 12 hours post-infection with type III interferon or with the synthetic viral RNA analog poly(I:C) [24,25]. Altogether, these studies demonstrate that stimulation of the antiviral innate immune response before infection or at the very early stage of infection inhibits SARS-CoV-2 replication and pathogenesis, as expected given the high level of SARS-CoV-2 sensitivity to prophylactic type I and type III IFN treatment observed in cell culture [2–4]. By contrast, our study provides the first evidence that administration of type I IFN as soon as the animals exhibited the first clinical signs, corresponding to weight loss, three days post-infection, was not associated with any change in clinical signs compared to placebo treated hamsters. This study thus does not support the use of intranasal type I IFN as a therapeutic in patients with COVID-19 symptoms.

In comparison to humans, virus replication and lung pathology progress much faster in hamsters, which have a peak of virus replication in the lungs at day 2–3 and strongest expression of clinical signs at day 6–7 [20]. Treatment at day 3 post-infection thus corresponds to a “late” time point for treatment initiation in hamsters. We detected an upregulation of IL-6, CCL2 and TNF-α protein levels in the IFN-late group, compared to the IFN-pre and IFN-early groups. However, this did not result in enhanced pathology compared to the placebo group. Our results therefore indicate that type I IFN treatment at late time-points is unlikely to be associated with deleterious immunopathology exacerbation mechanisms, which were observed in SARS-CoV-1 and MERS-CoV infected mice and feared in SARS-CoV-2-infected humans [13,15,16]. A contribution of type I IFNs to SARS-CoV-2 lung pathology has been suggested from work in IFNAR knockout mice transduced with adenovirus expressing human ACE2 and in STAT2 knockout hamsters [24,26,27]. However, heightened viral loads were also observed in IFNAR knockout mice and STAT2 knockout hamsters, illustrating the fact that type I IFNs can have beneficial and deleterious effects most likely depending on the stage of SARS-CoV-2 infection [24,27]. SARS-CoV-2 expresses a broad array of type I IFN signaling antagonists that likely account for the low sensitivity observed in post-infection type I IFN treatments in cell culture (S7 Fig), and for the lack of beneficial effects observed in this study when type I IFN treatment was initiated at the onset of symptoms three days post-infection [4,28–31].

Even though SARS-CoV-2 expresses a broad array of type I IFN signaling antagonists, we detected a significant upregulation of ISGs expression in the respiratory tract of SARS-CoV-2 infected hamsters, similarly to what has been described in COVID-19 patients [32]. Interestingly, ISG expression in the respiratory tract was not further increased by IFN treatment, as previously observed in MERS-CoV infected mice treated with IFN-beta at 2 days post-infection [16]. This result suggests that ISG levels had reached their maximal expression in response to virus-induced endogenous type I and type III IFNs production and could not be further augmented following exogenous type I IFN administration.

Our study demonstrates that the timing of the type I IFN treatment is critical for its efficacy in a preclinical model of severe SARS-CoV-2 infection. Results from the SOLIDARITY clinical trial showed no benefit of subcutaneous interferon-β-1a injection, while a phase-two clinical trial provided evidence of some benefits of inhaled interferon-β-1a in COVID-19 patients [12,33]. The route of type I IFN administration was not the sole difference between these trials, as the patients treated in the SOLIDARITY trial were on average at a more severe stage of the disease. Our findings support the hypothesis that type I IFN treatment may only be beneficial in patients with low viral load or with mild symptoms at the early stages of the disease, while it likely does not provide any benefit in COVID-19 patients requiring hospitalization [31,34].

Materials and methods

Ethics statement

The animal experimentation protocols complied with the regulation 2010/63/CE of the European Parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. These experiments were approved by the Anses/ENVA/UPEC ethic committee and the French Ministry of Research (Apafis n°24818–2020032710416319).

Animals and interferon-α treatment

We purchased (n = 78) eight week-old female Syrian golden hamsters (Mesocricetus auratus, strain RjHan:AURA) from Janviers’s breeding Center (Le Genest, St Isle, France) and housed them in an animal-biosafety level 3 (A-BSL3), with ad libidum access to water and food. Animals weighing on average 97 grams at 1 day before infection (dbi) were randomly assigned to five groups: 12 non-treated non-infected animals (NI), 18 placebo-treated infected animals (Placebo), 18 infected animals interferon (IFN)-α-treated at 16 hours before infection (hbi), 1 day post-infection (dpi) and 3 dpi (IFN-pre), 18 infected animals IFN-treated at 1 and 3 dpi (IFN-early), and 12 infected animals IFN-treated at 3 and 5 dpi (IFN-late).

Human equivalent dosing in hamsters was calculated based on the dosing for subcutaneous interferon-β-1a (12x106IU) and inhaled interferon-β-1a (6x106IU) in clinical trials in COVID-19 patients, corresponding to an average of 1.5x105IU/kg [12,33]. The human dose was multiplied by 7.4 in order to get the hamster equivalent dose of 106IU/kg or 105IU/hamster weighing on average 100g, based on body surface area [19].

At day 1, 3 and 5, all animals were anesthetized with isoflurane and treated by the intranasal route either with 150μL (75μL in each nostril) PBS (Placebo) or with 150μl (75μL in each nostril) PBS containing 105 IU of recombinant universal IFN-α (IFN) (Hu-IFN-αA/D[Bg/II], pbl assay science, Piscataway, NJ). Animals from group IFN-pre were also anesthetized and IFN-treated 1 day prior to infection. In a preliminary experiment designed to test the efficacy of recombinant universal IFN-α in Syrian hamsters, (n = 6) eight week-old female Syrian golden hamsters (Mesocricetus auratus, strain RjHan:AURA), purchased from Janviers’s breeding Center (Le Genest, St Isle, France) were treated by the intranasal route with 150μl (75μL in each nostril) PBS containing 105 IU IFN. At day 1 post-treatment the animals were euthanized to harvest tissues for gene expression analyses. In a third experiment, (n = 20) eight week-old (Mesocricetus auratus, strain RjHan:AURA), also purchased from Janviers’s breeding Center (Le Genest, St Isle, France), were either treated by the intranasal route with 150μL (75μL in each nostril) PBS (Placebo) or with 150μl (75μL in each nostril) PBS containing 105 IU IFN, or with 150μl (75μL in each nostril) PBS containing 7.105 IU IFN. Tissues were harvested either at day 1 or day 2 post-treatment, for gene expression and protein levels analysis.

Virus and experimental infection

SARS-CoV-2 strain UCN1 was amplified as described previously and used at passage 2 [35]. The viral stock was sequenced by Eurofins Genomics (Ebersberg, Germany) using the Illumina deep sequencing Eurofins Genomics Covid Pipeline v.0.1. Sequence analysis revealed that the virus had an intact spike cleavage site. Animals were anesthetized with isoflurane and intranasally inoculated with 104 TCID50 units of UCN1 SARS-CoV-2 strain split in 20μL in each nostril. Non-infected animals received the equivalent amount of PBS. Animals were weighted daily from 1 dbi to 15 dpi. Oro-pharyngeal swabs were performed daily from 1 dpi to 6 dpi and at 8, 10 and 12 dpi. Six animals from groups Placebo, IFN-pre and IFN-early were anesthetized and euthanized by exsanguination at 2 dpi and then necropsied. Six animals from each group were also necropsied at 5 dpi. All remaining animals were necropsied at 15 dpi. For each necropsied animal, the following samples were collected: EDTA whole blood, lungs, spleen and nasal turbinates. Organs were either stored frozen at -80°C in TRIzol reagent (Invitrogen, Carlsbad, CA) or Dulbecco’s Modified Eagle medium (DMEM) containing penicillin and streptomycin, or stored in 10% neutral formalin.

Histology

Samples from lung, upper trachea and larynx were fixed by immersion in 10% neutral-buffered formalin and processed routinely into paraffin wax. 4 μm sections were cut and stained with haematoxylin and eosin (H&E). In addition, samples were stained using the RNAscope in-situ hybridization (ISH) technique to identify the SARS-CoV-2 virus RNA, as previously described [36]. Briefly, tissues were pre-treated with hydrogen peroxide for 10 minutes (room temperature), target retrieval for 15 mins (98–101°C) and protease plus for 30 mins (40°C) (Advanced Cell Diagnostics). A V-nCoV2019-S probe [Cat No. 848561, Advanced Cell Diagnostics] was incubated on the tissues for 2 hours at 40°C. Amplification of the signal was carried out following the RNAscope protocol using the RNAscope 2.5 HD Detection kit–Red (Advanced Cell Diagnostics). Tissue sections were also stained using immunohistochemistry (IHC) to detect Mx1 using a monoclonal antibody (Merck Sigma-Aldrich MABF938 clone M143/CL143) at 1:1,000. Tissues were dewaxed before heat-induced epitope retrieval was performed using Leica ER1 (pH 6.0) solution for 20 minutes on the Leica Bond RXm automatic stainer. Tissues were treated with hydrogen peroxide (5 mins) and a universal blocker (Superblock TBS blocking buffer, Thermo Scientific; 15 mins) before incubating the antibody for 15 mins. The Leica Bond Polymer Refine detection kit was used for visualisation and counterstaining. Tissue slides were scanned with a Hamamatsu Nanozoomer S360 scanner, visualized with NDP.view2 software and examined by a veterinary pathologist blind to the experimental conditions. A semi-quantitative scoring system was applied to evaluate the lung lesions induced by SARS-CoV-2 as follows: 0 = no lesions; 1 = occasional airway epithelial degeneration and alveolar wall/space infiltration affecting up to 5% of the slide; 2 = mild airways epithelial cell degeneration and alveolar wall and space infiltration, multifocal affecting up to 25% of the slide with presence of peribronchiolar and perivascular cuffs, normally incomplete; 3 = moderate presence of airway epithelial cells degeneration and alveolar wall and space infiltration, multifocal affecting up to 50% of the slide with presence of abundant peribronchiolar and perivascular cuffs; 4 = marked presence of airway epithelial cells degeneration and alveolar wall and space infiltration, multifocal affecting more than 50% of the slide with presence of abundant large peribronchiolar and perivascular cuffs. Digital image analysis Nikon-NIS-Ar software (version 4.30.01) was used to calculate the percentage of area with lesion and with positive staining in ISH and IHC slides.

Hematology

For each necropsied animal, a complete blood count was performed within 15 minutes of sampling on a ProCyte Dx analyser (IDEXX laboratories, Westbrook, ME). Blood films were also performed, air-dried, stained with May-Grünwlad-Giemsa stain, fixed and coverslip-mounted. They were examined by a board-certified veterinary pathologist, blinded to the experimental conditions, to estimate the leukocyte differential count. The percentages of neutrophils, lymphocytes, monocytes, eosinophils and basophils were estimated from 100 cells. Samples with blood clots were excluded from the hematological analysis.

RNA extraction from tissue samples and cDNA synthesis

For each organ, 30 mg portions of tissue were placed in tubes with beads (Precellys lysis kit; Stretton Scientific, Ltd., Stretton, United Kingdom) filled with 500 μL of TRIzol reagent (Invitrogen, Carlsbad, CA) and mixed for 5 s at 6,000 rpm three times in a bead beater (Precellys 24; Bertin Technologies, Montigny-le-Bretonneux, France). After TRIzol extraction, the aqueous phase was transferred to 96-well plate and processed according to the manufacturer’s instructions (NucleoMag RNA; Macherey-Nagel GmbH & Co, Germany) using a KingFisher automated platform (Thermo Fisher Scientific, Inc., Ontario, Canada). cDNA was synthesized by reverse transcription of 500 ng of total RNA using both oligo(dT)18 (0.25 μg) and random hexamer (0.1 μg) and a RevertAid first-strand cDNA synthesis kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Quantitative PCR from tissue samples

Quantitative PCR for the analysis of host genes expression was performed in 96-well plates in a final volume of 10 μL on a LightCycler 96 (Roche, Mannheim, Germany). Mixes were prepared according to the manufacturer’s instructions (QuantiFast SYBR green PCR; Qiagen) with 1 μL of 1:2 diluted cDNA and a final 1 μM concentration of each primer (S1 Table). Relative quantification was carried out by using the 2-ΔCT method, and the geometric means of two housekeeping genes (RPL18 and RPS6KB1).

Quantitative PCR from oropharyngeal swabs

Viral RNA was extracted from 160 μL of oro-pharyngeal swabs stored in 500μL of DMEM with antibiotics. RNA extraction was performed by using Qiagen Viral RNA mini kit according to the manufacturer’s instructions (Qiagen, Les Ulis, France), with the addition of 15μL Triton X-100 (MP Biomedicals, Illkirch, France) to 560 μL of AVL Lysis buffer for each sample. TaqMan RT-qPCR was performed according to the manufacturer’s instruction (QuantiTect Probe RT-PCR; Qiagen) using primers targeting the envelope protein gene (E gene) and a previously described protocol [37]. Absolute quantification was performed using a standard curve based on six 10-fold dilutions of a quantitative Synthetic RNA from SARS-CoV-2 (BEI Resources: Catalog No. NR-52358).

IL-6 ELISA from lung samples

150 mg portions of lung tissue were placed in tubes with beads (Precellys lysis kit; Stretton Scientific, Ltd., Stretton, United Kingdom) filled with 1 mL of Dulbecco’s Modified Eagle medium (DMEM) and mixed for 5 s at 6,000 rpm three times in a bead beater (Precellys 24; Bertin Technologies, Montigny-le-Bretonneux, France). Supernatant was collected after a brief centrifugation and a commercial hamster IL-6 double antibody enzyme-linked immunosorbent assay kit was performed according to the manufacturer’s instructions (ELISA Genie, Dublin, Ireland).

Cytokine/Chemokine quantitation using a multiplex assay

150 mg portions of lung tissue were placed in tubes with beads (Precellys lysis kit; Stretton Scientific, Ltd., Stretton, United Kingdom) filled with 1 mL of Dulbecco’s Modified Eagle medium (DMEM) containing Triton X100 (1% v/v to inactivate the virus) and a protease inhibitor cocktail (Sigma). Tubes were mixed for 5 s at 6,000 rpm three times in a bead beater (Precellys 24; Bertin Technologies, Montigny-le-Bretonneux, France). Plasma samples also contained Triton X100 (1% v/v to inactivate the virus) and a protease inhibitor cocktail (Sigma). The custom Syrian Hamster Panel was developed by Merck-Millipore under the reference number SPRCUS1249, using previously identified cross-reactivity with the potential to detect hamster proteins from pre-developed commercial assays for rat (RECYTMAG-65K) and feline (FCYTMAG-20K) species, respectively. It was used for the measurement of IL-1β, CCL2, TNFα, CXCL10, and IL-10. The custom Syrian Hamster Milliplex xMAP kit (SPRCUS1249, Merck-Millipore) is available upon request to the corresponding author. Data were recorded on a MagPix instrument using Xponent software (Luminex). Results are expressed as concentration in pg/mL. Among the cytokines and chemokines included in the multiplex assay, we detected a robust signal for CXCL10, IL-10, IL-1β in lung and plasma samples. However, CCL2 and TNF-α were only detected in plasma samples, most likely due to higher signal to noise ratio in plasma samples than in lung samples.

In vitro IFN-α treatment

Vero-E6 cells were grown in DMEM containing 1% antibiotics (penicillin/streptomycin) and 10% fetal bovine serum. Cells were treated with 1000 UI/mL recombinant universal IFN-α (IFN) (Hu-IFN-αA/D[Bg/II], pbl assay science, Piscataway, NJ) for 18 hours prior to infection and were subsequently infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 10−3. Infections were carried out in DMEM containing 1% antibiotics (penicillin/streptomycin) and 2% fetal bovine serum. Recombinant universal IFN-α (Hu-IFN-αA/D[Bg/II], pbl assay science, Piscataway, NJ) was added at a final concentration of 1000 UI/mL to the medium 6 hours post-infection for cells treated subsequently to the infection. Culture supernatants were harvested 24 hours post-infection and viral titers were determined by the TCID50 method on Vero-E6 cells and calculated by the Spearman & Kärber algorithm.

SARS-CoV-2 titrations from lung samples

Titrations were performed on 90% confluent Vero-E6 cells in 96-well plates. Viral titers were calculated using the Spearman-Kärber method.

Statistical analyses

All the data, except weight loss and H&E lesion scoring, were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (GraphPad Prism Software, USA) with the p values indicated corresponding to the results of Tukey’s post hoc tests. Weight loss was statistically analyzed using two-way ANOVA with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test (GraphPad Prism Software, USA) with the p values indicated for the weight loss corresponding to the results of Tukey’s post hoc tests. H&E lesion scores were statistically analyzed using the Mann-Whitney test.

Supporting information

S1 Table. List of primers used in this study.

(DOCX)

Click here for additional data file. (46.8KB, docx)
S1 Fig. Subgenomic viral RNA in nasal turbinates.

Nasal turbinates were harvested at day 2 post-infection (D2) or day 5 post-infection (D5). Viral sgRNA levels relative to the housekeeping genes RPL18 and RPS6KB1 were determined by RT-qPCR. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (194.3KB, tif)
S2 Fig. Histological analysis of the impact of IFN-α treatments.

Representative pictures were selected to display the pathology from haematoxylin and eosin (H&E) stained lung section, viral RNA in lung sections stained with RNAScope in situ hybridization (ISH) and Mx1 protein detected by immunohistochemistry (IHC). D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Scale bar: 100μm.

(TIF)

Click here for additional data file. (5.2MB, tif)
S3 Fig. Type I IFN-α treatment does not prevent lymphocytopenia.

A complete blood count analysis was performed as described in the methods section. D2: day 2 post infection; D5: day 5 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (200.5KB, tif)
S4 Fig. Impact of type I IFN-α treatment on the immune response to SARS-CoV-2. in the lung.

Lung transcripts levels of IL-1β, IL-12, IFN-α7, OAS3, Mx2, CCL5 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (1MB, tif)
S5 Fig. Impact of IFN-α treatment on the immune response to SARS-CoV-2 in the nasal turbinates.

Nasal turbinates transcripts levels of Mx1, Mx2, ISG15, IFN-α7, CXCL10, CCL5, IL-1β, IL-6, IL-10, IFN-γ and IL-12 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (2MB, tif)
S6 Fig. Impact of IFN-α treatment on the immune response to SARS-CoV-2. in the spleen.

Spleen transcripts levels of Mx1, Mx2, ISG15, IFN-α7, CXCL10, CCL5, IL-1β, IL-6, IL-10, IFN-γ and IL-12 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (1.8MB, tif)
S7 Fig. Effect of IFN-α treatment on SARS-CoV-2 replication in cell culture.

Vero-E6 cells were treated either with placebo or with 103 UI/mL recombinant universal IFN-α 18 hours prior to infection (IFN-pre) or 6 hours post infection (IFN-post). Equivalent volume of PBS was used as a negative control. Viral titers were determined by TCID50 from supernatants collected 24 hours post infection. Each dot represents a technical replicate of a representative experiment performed twice. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

(TIF)

Click here for additional data file. (62.3KB, tif)

Acknowledgments

We thank Meriadeg Ar Gouilh and Astrid Vabret (University Hospital of Caen, Normandie Université, Caen, France) for granting access to the SARS-CoV-2 UCN1 strain and Daniel Gonzalez-Dunia for proofreading the manuscript. We thank Vanessa Bastid, Mélanie Badré-Biarnais, Jean-Marc Boucher, Anouck Labadie, Carine Peytavin de Garam, Jonathan Rieder and Jean-Luc Schereffer for their investment in virological and serological analyses; Valère Brogat, Sébastien Kempff and Estelle Litaize for animal care and experimentation (Nancy laboratory for rabies and wildlife, ANSES, Lyssavirus Unit, Malzéville, France).

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was funded by a grant from the Agence Nationale de la Recherche (ANR-20-COV5-0004) to RV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Michael S Diamond

26 Mar 2021

Dear Dr. Volmer,

Thank you very much for submitting your manuscript "Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Note that the reviews were mixed with one recommending rejection in large part based on novelty due to the published paper by the TenOever laboratory. The other reviewers and I feel there are some additional interesting (although not completely clear) results that could make this paper a sound addition to the SARS-CoV-2 field. In the revision, you will need to address the following: (a) make sure key experiments have independent biological repeats at separate times; (b) provide greater information and methods on the cytokine assay; (c) perform additional ISH and histological analysis as suggested by the Reviewers and (d) provide more discussion as to the lack of antiviral effect of IFN in some of the experiments and how this relates to prior studies.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael Diamond

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Bessière et al evaluated the potential therapeutic effects of high dose type I interferon treatment against SARS-CoV-2 infection in the Syrian hamster model. One day before or after infection, high dose IFN treatment had a modest effect on weight loss. Starting IFN treatment 3 dpi, no effect was observed. Surprisingly, pre-treatment with type I IFN had no effect on infectious virus titer and viral RNA load. In addition, no significant difference in lung pathology was observed in the pre-treated animals, albeit this may be due to the relatively small number of animals per group. Initiating IFN treatment one day after infection had significant effects on lung pathology 2 dpi, and reduced viral RNA load and infectious virus titer 5 dpi. No effect on weight loss, inflammation or virus titer was observed in hamsters treated with IFN starting 3 dpi. Combined these data are rather surprising and it poses the question, why does type I IFN not protect hamsters from SARS-CoV-2 challenge. The authors demonstrate increased Mx2 gene expression following treatment, suggesting that the universal type I IFN is able to induce an antiviral immune response. Another possibility is that the antiviral immune response is not effective against SARS-CoV-2, or the antiviral immune response is not induced in the target cell of SARS-CoV-2. To distinguish between the two possibilities, the authors should perform their Mx1 RNA-ISH staining on interferon treated animals without SARS-CoV-2 challenge. This allows the authors to provide additional insight as to why type I IFN provides minimal protection and compare the antiviral response between treatment and actual infection. Finally, the authors present some intriguing cytokine and chemokine protein data by ELISA and multiplex array on hamster lung homogenates and sera. If these were available to the general scientific community, this would be a major advance for the field. However, minimal details are provided in terms of product number, antibody clones etc. A search for Syrian Hamster cytokines MilliPlex xMAP assay on the company website yields no hits. AssayGenie appears to have several ELISA kits including IL-6, IL-1b etc., but there is no mention of CXCL10 or CCL2. Additional information must be provided to allow others to make use of these reagents and assays.

Major comments:

• All experiments appear to have been done in one large experiment. The authors should consider repeating key findings in a second experiment.

• Details on the ELISA or bead-array kits are missing. What kind of

• RNA-ISH using Mx1 on IFN treated animals will provide important insight into the reason for the lack of a robust effect of type I IFN administration prior to virus challenge.

• Please discuss the surprising lack of IFN effect on virus titers and inflammation in this model.

Minor:

• Type in the methods section on the hamsters. It appears they are 78 weeks old….probably 7-8 weeks old.

• Provide some more details in the “main text” such as the source and dose the type I IFN used. This can be found in the materials and methods section, but should be referenced elsewhere.

Reviewer #2: ‘Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model’ by Bessière et al. examines the impact of intranasal delivery of universal IFNa to SARS-CoV-2 infected hamsters in either prophylactic or therapeutic treatment models. The article is well written and contains appropriate citations of the recent literature. While much of the data from this manuscript confirms previously published works, new details concerning the timing of treatment are revealed in this manuscript and are important to the field. The most dramatic differences in treatment result appear to be in lung histopathology; it would greatly improve the manuscript if this data was expanded upon with high magnification images and characterization of immune cell infiltrates. Overall, this paper demonstrates modest efficacy of type I IFN treatment in preventing the most serious signs of disease including reduced viral titer in the lung, improved lung histopathology and reduction in some cytokine/chemokine levels. However given the compressed treatment window in small animal models, these results are still significant and applicable to COVID-19 patient treatment.

Reviewer #3: Bessiere et al. use the hamster model of SARS-CoV-2 infection to test the efficacy of IFNa treatment on infection on outcome regarding lung viral loads and pathology. For us it not clear where the novelty of the results are, in particular as very similar data have been presented earlier in the same model (Hoagland et al. Immunity 2021) yet in much greater detail and trialing many more conditions (dose response, full DEG analysis). Furthermore, the authors fail to provide strong evidence for a a clear benefit from an proposed IFN regimen. Obviously hamsters constitute a very stringent challenge model, however, it had been demonstrated before that efficient therapeutic interventions for SARS-COV-2 are in principle possible in hamsters (e.g. Kaptein et al. PNAS 2020, in that case using RdRP inhibitors) and hence a --if at all-- 1log reduction in viral loads and almost no reduction in any of the measured cytokines may not be considered an incremental gain in knowledge towards promising treatment options.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Key findings should be repeated in a second cohort of animals

RNA-ISH in Mx1 in IFN treated animals to demonstrate the location and intensity of the antiviral host response.

Reviewer #2: The most significant and consistent results showing an effect of IFN treatment were in the ‘early’ treatment group. Do the authors think prophylactic treatment would have been more effective if daily dosing was used instead of every 2 day dosing? How long does ISG stimulation last after treatment? Prophylactic treatment results are not strong as the Hoagland et al, Immunity paper.

Figure 2A – Figure 2 shows the strongest protection data in the manuscript and more detail to explain these results is key to demonstrating the utility of IFN treatment. Why are the lung sections shown from D5 post infection when there are not significant differences in histology score? Additional, high magnification, histopathology images should be included.

The authors should discuss why titer changes are observed only in the lower airway when strong ISG induction was observed in nasal turbinates.

Reviewer #3: Dose response for (i) virus inoculum and (ii) interferon for infection outcome.

The quantification of lung areas/volumes affected may need more vigorous means for quantification to tease out differences, if at all.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: Figure 1B – Is weight loss significantly different between IFN treated and sham treated groups of hamsters?

It would be very helpful if the titer data in figure 1C was labeled as being from oralpharangeal swabs and D/E being from the lung.

Figure 2B – significance notations are confusing and don’t seem to match the text. Please clarify if all treatment conditions are significant or if only IFN-early led treatment to significant reductions in histopathology.

Reviewer #3: The luminex assays for hamster cytokines are not described but may be of interest for he general public.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Decision Letter 1

Michael S Diamond

28 Jun 2021

Dear Dr. Volmer,

Thank you very much for submitting your manuscript "Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. There are just a few remaining editorial and data presentation comments that need to be addressed.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael Diamond

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The reviewers have analyzed the Mx1 gene and protein expression in hamsters after IFN treatment. This is very nice data and suggests that SARS-CoV-2 can infect and replicate in cells despite the presence of an antiviral immune response. What they have not done is repeat some of the key findings as requested. While I agree with the reviewers that some P-values are unlikely to change, such as the cytokine induction over mock, others certainly can. For example, the virus titer differences (Fig 1) on day 5 are barely significant.

Reviewer #2: ‘Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model’ by Bessière et al. examines the impact of intranasal delivery of universal IFNa to SARS-CoV-2 infected hamsters in either prophylactic or therapeutic treatment models. The article is well written and contains appropriate citations of the recent literature. While much of the data from this manuscript confirms previously published works, new details concerning the timing of treatment are revealed in this manuscript and are important to the field. The most dramatic differences in treatment result are in reduced weight loss, improved lung histopathology and a reduction in virus titer in the lung. High magnification images and generally improved data presentation have strengthened this manuscript. Overall, this paper demonstrates modest efficacy of type I IFN treatment in preventing the most serious signs of disease including reduced viral titer in the lung, improved lung histopathology and reduction in some cytokine/chemokine levels. However, given the compressed treatment window in small animal models, these results are still significant and applicable to COVID-19 patient treatment. Most importantly, the authors do not overstate their findings and accurately present them as part of our growing understanding of how to treat COVID-19 patients.

Reviewer #3: I may have to excuse for any possible oversight, maybe partially due to a lack of obvious mark-ups in the revised version of the manuscript available to me, however, it is not very clear to what extent and where the authors provide significant new insight in the revised version of the manuscript. The impression prevails that little efforts was made to consolidate previous claims. As a minimal requirement, the wording in title (now claiming a true benefit from IFN treatment) may need to be tempered. E.g. from “is beneficial” to “might confer some benefit” or similar. Here also stating “lack of therapeutic benefit of intranalsal IFN” may be appropriate and as such an important finding.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: My questions and concerns have been adequately addressed in this revised manuscript.

Reviewer #3: As mentioned before, the importance of the rather small differences regarding any of the parameters measured in infected hamsters (weight evolution, virus loads, RT-qPCR, histology score, cytokine) remains of concern. With this regard, I convene with the Editor and Reviewer #1 that consolidation of data in independent biological repeats may be required, at least for some selected key data; I have the impression that all data on infection outcome were generated from a single batch of animals. I understand the ethical consideration that aims to reduce animal suffering. However, if the authors claim (see line 180-181) that their study has relevance to change clinical practice, independent confirmation should be considered more seriously, including from an ethical point of view either endorsing or not to treat patients that are at high risk of developing severe disease.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The H&E score in Fig 2 is analyzed with an ANOVA. However, the data appears non-parametric and not normally distributed (day 15, early IFN-a). Please check statistical analysis

Reviewer #2: (No Response)

Reviewer #3: The quantitative scoring for histopathology may be accepted, fully trusting the expert pathologist’s view, however more details on the microscopic parameters and lesions quantified would be appreciated to level up with other studies using the same hamster model. Likewise, regarding my original request for more information on the novel cytokine multiplex assay, I understood the journal’s policy to enforce open science. The multiplex assay used here may be of quite some interest to the field that is in desperate need of new state-of-the-art research tools. More efforts in describing the performance, validation and technical specifications (e.g. list of reagents) of these tools would be very much appreciated.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Decision Letter 2

Michael S Diamond

7 Jul 2021

Dear Dr. Volmer,

We are pleased to inform you that your manuscript 'Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model' has been provisionally accepted for publication in PLOS Pathogens.

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PLOS Pathogens

Michael Diamond

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):

Acceptance letter

Michael S Diamond

4 Aug 2021

Dear Dr. Volmer,

We are delighted to inform you that your manuscript, "Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model," has been formally accepted for publication in PLOS Pathogens.

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Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. List of primers used in this study.

    (DOCX)

    Click here for additional data file. (46.8KB, docx)
    S1 Fig. Subgenomic viral RNA in nasal turbinates.

    Nasal turbinates were harvested at day 2 post-infection (D2) or day 5 post-infection (D5). Viral sgRNA levels relative to the housekeeping genes RPL18 and RPS6KB1 were determined by RT-qPCR. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (194.3KB, tif)
    S2 Fig. Histological analysis of the impact of IFN-α treatments.

    Representative pictures were selected to display the pathology from haematoxylin and eosin (H&E) stained lung section, viral RNA in lung sections stained with RNAScope in situ hybridization (ISH) and Mx1 protein detected by immunohistochemistry (IHC). D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Scale bar: 100μm.

    (TIF)

    Click here for additional data file. (5.2MB, tif)
    S3 Fig. Type I IFN-α treatment does not prevent lymphocytopenia.

    A complete blood count analysis was performed as described in the methods section. D2: day 2 post infection; D5: day 5 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (200.5KB, tif)
    S4 Fig. Impact of type I IFN-α treatment on the immune response to SARS-CoV-2. in the lung.

    Lung transcripts levels of IL-1β, IL-12, IFN-α7, OAS3, Mx2, CCL5 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (1MB, tif)
    S5 Fig. Impact of IFN-α treatment on the immune response to SARS-CoV-2 in the nasal turbinates.

    Nasal turbinates transcripts levels of Mx1, Mx2, ISG15, IFN-α7, CXCL10, CCL5, IL-1β, IL-6, IL-10, IFN-γ and IL-12 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (2MB, tif)
    S6 Fig. Impact of IFN-α treatment on the immune response to SARS-CoV-2. in the spleen.

    Spleen transcripts levels of Mx1, Mx2, ISG15, IFN-α7, CXCL10, CCL5, IL-1β, IL-6, IL-10, IFN-γ and IL-12 relative to the housekeeping genes RPL18 and RPS6KB1 determined by RT-qPCR. D2: day 2 post infection; D5: day 5 post infection; D15: day 15 post infection. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (1.8MB, tif)
    S7 Fig. Effect of IFN-α treatment on SARS-CoV-2 replication in cell culture.

    Vero-E6 cells were treated either with placebo or with 103 UI/mL recombinant universal IFN-α 18 hours prior to infection (IFN-pre) or 6 hours post infection (IFN-post). Equivalent volume of PBS was used as a negative control. Viral titers were determined by TCID50 from supernatants collected 24 hours post infection. Each dot represents a technical replicate of a representative experiment performed twice. Results are expressed as means ± SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test.

    (TIF)

    Click here for additional data file. (62.3KB, tif)
    Attachment

    Submitted filename: Reply to reviewers.docx

    Click here for additional data file. (56.9KB, docx)
    Attachment

    Submitted filename: Reply to reviewers round #2.docx

    Click here for additional data file. (47.7KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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