Study design

The primary goal of this study was to compare the prophylactic and therapeutic efficacy of RDV with the combination of LPV/RTV and IFNb. First, we assessed antiviral efficacy and cytoxicity in the Calu-3 human lung cell line as compared with the appropriate vehicle control. Experimental conditions in vitro were performed in sextuplicate unless otherwise stated, and antiviral assays were repeated four times per drug. Second, we evaluated the in vivo efficacy of prophylactic RDV as compared to vehicle with two different doses of MERS-CoV in a new transgenic mouse model of MERS-CoV pathogenesis with improved pharmacokinetics for nucleotide prodrugs. We performed two additional prophylactic studies comparing RDV and vehicle to LPV/RTV-IFNb, IFNb-alone, and their vehicles. Third, we assessed the therapeutic efficacy of the above treatment regimens in a mouse model of MERS-CoV pathogenesis, but did not include an IFNb, only arm and these studies were performed twice. All lung histological assessments were performed in a blinded manner. In addition, we performed a single therapeutic efficacy study with a lethal dose of MERS-CoV. Our in vivo efficacy studies were designed to mirror the ongoing MIRACLE human clinical trial which is evaluating combination LPV/RTV-IFNb. Our studies were intended to generate the data required to justify further testing in nonhuman primates and collectively inform future human clinical trials. Mice were age- and sex-matched and randomly assigned into groups before infection and treatment. Exclusion criteria for in vivo studies were as follows: If a given mouse unexpectedly did not lose weight after infection and their virus lung titers were more than 2 log 10 lower than the mean of the group, this indicated that infection was inefficient, and all data related to that mouse were censored.

Animal care and ethics statement

Efficacy studies were performed in animal biosafety level 3 facilities at UNC Chapel Hill. All works were conducted under protocols approved by the Institutional Animal Care and Use Committee at UNC Chapel Hill (IACUC protocol #16-284) according to guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.

Virus

For in vitro studies, a MERS-CoV reporter virus expressing nanoluciferase was employed (MERS-nLUC)17. MERS-nLUC stocks were derived from a molecular clone through electroporation of Vero 81 cells (ATCC CCL-81) and isolation of virus through harvesting of culture supernatants53. Briefly, DNA fragments A–F encoding MERS-nLUC cDNA genome were ligated to create full-length cDNA, which was then used as a template for in vitro transcription. Full-length genomic RNA was then electroporated into Vero-81 cells yielding recombinant virus stock. The resultant stock was passaged twice in Vero 81 to generate a working stock (1.6E + 07 pfu/mL) for our studies. Wild-type MERS-CoV for comparative antiviral efficacy studies was derived from our EMC 2012 infectious clone as described above to obtain a working stock with a titer of 3E + 07 pfu/mL53. For in vivo studies, we utilized mouse adapted MERS-CoV passage 35 clone 4 (MERS M35C4)24. MERS M35C4 has 12 amino acid coding changes as well as a single-nucleotide change in the 5ʹ UTR and a large deletion in ORF4b/ORF5. This virus is a clonal isolate generated through serial passage of MERS-CoV in mice. After 35 passages in mice, virus was plaque purified and clone 4 was expanded two times on Vero CCL81 cells to obtain our working stock. The working stock (1.1E + 08 pfu/mL) was created in virus collection medium (Optimem (Gibco), 3% Fetal Clone II serum product (Hyclone), and antibiotic/antimycotic (Gibco) and non-essential amino acids (Gibco)).

Compounds and formulation for in vitro studies

Remdesivir (RDV), lopinavir (LPV), and ritonavir (RTV) were solubilized in 100% DMSO and provided by Gilead Sciences, Inc. Recombinant human interferon beta (IFNb) protein was purchased from R&D Systems (8499IF010/CF, 2.8 × 108 IU/mg compared with WHO standard) and solubilized in sterile water as recommended.

In vitro efficacy and cytotoxicity in Calu-3 cells

To determine if virus replication kinetics and RDV susceptibility were similar among wild-type (WT) MERS-CoV EMC 2012 and MERS-nLUC, we performed comparative antiviral assays the human lung epithelial cell line, Calu-3 2B4 (kindly provided by Dr. Chien Tseng University of Texas Medical Branch). Calu-3 2B4 was maintained in the DMEM (Gibco), 20% fetal bovine serum (FBS, Hyclone), and 1× Antibiotic–Antimycotic (A/A, Gibco). Briefly, 48 h prior to infection, Calu-3 cells were plated at 4.3E + 04 cells/well. Twenty-four hours prior to infection, culture medium was exchanged with fresh medium. Calu-3 2B4 cells were infected with WT EMC 2012 or MERS-nLUC at a multiplicity of infection (MOI) of 0.1 for 1 h at 37 °C after which cells were washed, and a dose response of RDV diluted in twofold steps in media (DMEM, 10% FBS, DMEM, 1x A/A) was added in duplicate. Cells were then incubated at 37 °C in 5% CO 2 for 24 h after which 100 µl of media from each well was collected and assayed for virus production by plaque assay in Vero CCL81 cells. Briefly, 500,000 Vero CCL81 cells/well were seeded in six-well plates. The following day, medium was removed, and serial dilutions of sample were added per plate (10−1–10−6 dilutions) and incubated at 37 °C for 1 h, after which wells were overlayed with 1× DMEM, 5% Fetal Clone 2 serum, 1× A/A, 0.8% agarose. Three days after, plaques were enumerated to generate a plaque/ml value. The IC 50 value was defined in GraphPad Prism 7 (GraphPad).

To better understand the antiviral activities of RDV, LPV, RTV, IFNb, or combinations of LPV/RTV or LPV/RTV/IFNb, we performed efficacy and cytotoxicity assays in human lung epithelial cells (Calu-3). Cells were plated as described above, and infected with MERS-CoV expressing nanoluciferase (MERS-nLuc) in sextuplicate at an MOI of 0.08 for 1 h in the presence of a dose response of drug as described below. After 1 h of infection, virus was removed, cultures were rinsed once with medium, and fresh medium was added containing dilutions of drug. RDV (i.e., GS-5734, stock at 20 mM) was serially diluted in 100% DMSO in twofold increments to obtain a ten-point dilution series. Human IFNb (R + D Systems, 200 µg/mL or 5.6 × 107 IU/mL) was similarly diluted in PBS. LPV (10 mM stock) and RTV (10 mM stock) were similarly diluted in DMSO, although various amounts of stock compound were added directly to media to obtain the top four dilutions in the ten-point curve (50 µM, 25 µM, 12.5 µM, 6.25 µM). To model the antiviral effect of the fixed dose combination of LPV/RTV used to treat HIV, we performed antiviral assays with a fixed combination of LPV/RTV (weight:weight ratio of 4:1 or molar ratio 4.6:1). The LPV/RTV combination was serially diluted in 100% DMSO in twofold increments, but mixture stock was added directly to the media similar to above to obtain the top four concentrations. These studies were repeated in 4 independent experiments.

To model LPV/RTV in combination with IFNb, we first performed comparative equilibrium dialysis (CED)23 and determined that 5 µM LPV in 10% FBS containing cell culture medium gave the same amount of free (i.e., unbound to protein) LPV as the maximum concentration attained in human plasma (C max , i.e., 15 µM). We then combined a dose response of human IFNb with a fixed concentration of LPV/RTV (molar ratio of 4.6:1, (5 µM Lopinavir and 1.1 µM Ritonavir) based on the human plasma equivalent maximal concentration of LPV as determined by CED (i.e., 5 µM). Thus, this study was aimed at determining if the maximal amount of LPV attainable in humans provided an additive or synergistic antiviral effect when combined with IFNb. All studies were performed in cell culture medium containing 10% FBS. DMSO (0.5%) was constant in all conditions. At 48 h post infection (hpi), virus replication was quantified on a Spectramax (Molecular Devices) via nanoluciferase assay (NanoGlo Promega). Values from replicate wells per condition were averaged and compared with controls to generate a percent inhibition value for each drug dilution. The IC 50 value was defined in GraphPad Prism 7 (GraphPad) as the concentration at which there was a 50% decrease in viral replication using UV-treated MERS-nLUC (100% inhibition) and vehicle alone (0% inhibition) as controls. To measure cytotoxicity, cells were exposed to the same drug dilutions and controls as the efficacy studies, but in the absence of virus infection. After 48 h of exposure, cell viability was determined by Cell-Titer-Glo Assay (Promega) and quantitated on a Spectramax. Similar data was obtained in at least three independent experiments.

Formulations for in vivo studies

RDV was solubilized at 2.5 mg/ml in vehicle containing 12% sulfobutylether-β-cyclodextrin sodium salt in water (with HCl/NaOH) at pH 5.0. LPV (32 mg/mL) RTV (8 mg/mL) was solubilized in vehicle containing 90% propylene glycol and 10% ethanol. The ratio of LPV to RTV was held at 4:1 (weight:weight) for all studies herein. Recombinant mouse IFNb protein was purchased from R&D Systems (8234-MB/CF, 1.2 × 109 IU/mg calibrated against Murine IFN-beta WHO International Standard) for the in vivo studies and reconstituted in PBS.

Animals

MERS-CoV binds the human receptor dipeptidyl peptidase 4 (DPP4) to gain entry into cells, and two residues (288 and 330) in the binding interface of the mouse ortholog prevent infection of mice. We recently developed a mouse model for MERS-CoV through the mutation of mouse DPP4 at 288 and 330 via CRISPR/Cas9 thus humanizing the receptor (hDPP4) and rendering mice susceptible to MERS-CoV infection22. The serum carboxylesterase 1c (Ces1c) in mice drastically reduces RDV stability. Thus, all in vivo studies must be performed in mice genetically deleted for Ces1c (Ces1c−/−, stock 014096, The Jackson Laboratory)17. In order to perform in vivo efficacy studies with RDV and MERS-CoV in mice, we generated humanized DPP4 mice deficient in Ces1c expression (C57BL/6J Ces1c−/− hDPP4, The Jackson Laboratory stock number 403188). Briefly, our C57BL/6J hDPP4 mouse was rederived using Ces1c−/− oocytes. The resultant heterozygous mice were crossed with Ces1c−/− mice to generate mice homozygous for the Ces1c deletion (Ces1c−/−) and heterozygous for the hDPP4 alleles. The resultant offspring were genotyped and bred to generate founders homozygous for both Ces1c−/− and hDPP4.

MERS-CoV pathogenesis in Ces1c −/− and hDPP4 mice

To determine if the pathogenesis of MERS-CoV in Ces1c−/− and hDPP4 was similar to the parental hDPP4 line, we performed pathogenesis studies in the newly created Ces1c−/− and hDPP4 line. Similar numbers (N = 9–10/sex/group) of 23–24-week-old male and female mice were randomly assigned to each infection group. Mice were anaesthetized with a mixture of ketamine/xylazine and then intranasally infected with either 5E + 04 or 5E + 05 pfu MERS M35C4 in 50 µl virus collection medium. To monitor morbidity, mice were weighed daily out to 6 dpi. Mice that lost >20% of their starting weight were humanely sacrificed by isofluorane overdose. To better understand the magnitude of MERS-CoV replication and the typical metrics of disease, we infected 12 9–12-week-old female mice with 5E + 04 MERS M35C4 in 50 µl virus collection medium as done above. To monitor morbidity, mice were weighed daily. A subset of each cohort was randomly assigned for pulmonary function measurements by whole-body plethysmography (WBP, Data Sciences International) daily28. On 6 dpi, animals were killed by isoflurane overdose, lungs were scored for lung hemorrhage, and the inferior right lobe was frozen at −80 °C for viral titration via plaque assay as described above22. Lung hemorrhage is a gross pathological phenotype readily observed by the naked eye driven by the degree of virus replication where the coloration of the lung changes from pink to dark red25,26. The large left lobe was placed in 10% buffered formalin and stored at 4 °C for 1–3 weeks, until histological sectioning and analysis. Lung sectioning, hematoxylin and eosin staining as well as MERS-CoV antigen (primary antibody 1:500, sera from mice vaccinated with MERS-CoV nucleocapsid antigen) staining was performed by the Animal Histopathology & Laboratory Medicine Core at UNC.

Interferon beta pharmacodynamic studies

To generate a pharmacokinetic and pharmacodynamic (PK/PD) relationship for IFNb, we subcutaneously administered mouse IFNb (R + D Systems) to 18–20 week-old male and female Ces1c−/− or Ces1c−/− hDPP4 mice. Human equivalent dosing in mice was calculated based on the recommended dosing strategy for Betaseron (human IFNb, 0.13 million international units (MIU)/kg every other day)54. The human dose was multiplied by 12.3 in order to get the mouse equivalent dose of 1.6 MIU/kg or 4.8E4 IU/30 g mouse (40 µg/30 g mouse) based on body surface area55. First, we compared 1× and 2.5× human equivalent doses of IFNb in cohorts (N = 20/group) of Ces1c−/− mice. At 2, 4, 8, and 12 h post treatment, five mice per group were humanely euthanized, and plasma was snap-frozen at −80 °C, and peripheral blood mononuclear cells (PBMC) were isolated, place in Trizol LS (Thermo Fisher) and stored at −80 °C until analysis. Second, we compared the responses to a 25× human equivalent dose of IFNb (i.e., 40 MIU/kg or 1.2 MIU/30 g mouse (1000 µg/30 g mouse)) in Ces1c−/− or Ces1c−/− hDPP4 mice (N = 25/group) to ensure that these mouse lines respond similarly. At 0, 2, 4, 8, and 12 h post treatment, mice were humanely euthanized and plasma and PBMCs were isolated and stored as noted above. In addition, lungs were isolated at 4 and 8 h post treatment and stored in RNAlater (Thermo Fisher) at −80 °C until analysis. The total RNA was isolated from PBMCs using Zymo Research Direct-Zol RNA mini kit. Lung tissue was homogenized in Trizol (Thermo Fisher), and the total RNA was isolated similarly to PBMCs. The response to IFNb treatment in PBMCs was quantitated by qRT-PCR for the classic interferon stimulated gene MX dynamin-like GTPase 1 (Mx1) by TaqMan assay (Thermo Fisher Mm00487796_m1) using the Taqman Fast Virus 1-Step Master Mix (Thermo Fisher). Mx1 gene expression was compared with signals from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Thermo Fisher, Mm99999915_g1) and fold change over mock or time 0 post treatment was calculated by the ∆∆Ct method56. To monitor induction of interferon induced protein expression, we quantitated levels of mouse IFN-gamma inducible protein 10 (IP10, CXCL-10) in plasma via ELISA (Invitrogen).

Prophylactic in vivo efficacy studies

For all drug studies, mice that lost >20% of their starting weight were weighed twice and subjected to an additional visual check each day for clinical signs (hunching, ease of mobility, lethargy, etc.). Mice that fell <70% of their starting weight were immediately euthanized. Mortality was defined as unexpectedly finding mice dead in the cage.

Several prophylactic studies were performed to determine if drug regimens could affect virus replication and/or disease progression. In the initial study, we only compared RDV and vehicle. Briefly, groups of 9–12-week-old male and female Ces1c−/− hDPP4 were randomly assigned to groups (n = 7–9) and acclimated for 5–7 days at biosafety level 3 (BSL3). Treatment with vehicle (see above) or RDV (25 mg/kg, BID subcutaneously) was initiated the day prior to infection. For MERS-CoV infection, mice were anaesthetized with a mixture of ketamine/xylazine and then intranasally infected with either 5E + 04 or 5E + 05 pfu MERS M35C4 in 50 µl virus collection medium. To monitor morbidity, mice were weighed daily. On 4 or 6 dpi, animals were killed by isoflurane overdose, lungs were scored for hemorrhage (described above), and the inferior right lobe was frozen at −80 °C for viral titration via plaque assay as described above22. The large left lobe was placed in 10% buffered formalin and stored at 4 °C for 1–3 weeks, until sectioning and histological analysis. Lung sectioning, hematoxylin and eosin staining, as well as MERS-CoV antigen staining as described above were performed by the Animal Histopathology & Laboratory Medicine Core at UNC. Due to increased variability of infection and outcomes in male mice, all following studies were performed in only female mice.

We then performed two prophylactic studies to ascertain whether LPV/RTV-IFNb or IFNb alone could affect virus replication or disease progression. The studies were designed to mirror therapeutic efficacy studies. In the first study, in randomly assigned groups (n = 9–10) of 11–13-week-old Ces1c−/− hDPP4 female mice, we compared vehicle, LPV/RTV-IFNb, or IFNb alone. All treatments were initiated one day prior to infection. For MERS-CoV infection, mice were anaesthetized with a mixture of ketamine/xylazine and then intranasally infected with either 5E + 04 pfu MERS M35C4 in 50 µl virus collection medium. A human equivalent dose of a coformulation of LPV (160 mg/kg) and RTV (40 mg/kg) at 5 mL/kg was administered once daily via oral gavage. Groups receiving a 1× human equivalent dose of IFNb (R + D Systems, 1.6 MIU/kg or 4.8E4 IU/30 g mouse (40 µg/30 g mouse)) were dosed every other day via subcutaneous injection. To control for potential vehicle effects in the LPV/RTV-IFNb groups, we administered oral vehicle (90% propylene glycol and 10% ethanol) daily and subcutaneously injected with PBS every other day. RDV (25 mg/kg) at 10 mL/kg was administered twice daily via subcutaneous injection. As a control for RDV, an additional group was given subcutaneous vehicle. Mice were weighed daily to monitor morbidity. A subset (n = 4) of each cohort was randomly assigned for pulmonary function measurements by whole-body plethysmography (WBP, Data Sciences International) daily28. On 2 dpi, three animals per group were killed by isofluorane overdose, lungs were scored for hemorrhage (described above), and the large left lobe was frozen at −80 °C for viral titration via plaque assay as described above22. On 6 dpi, animals were killed and processed as done on 2 dpi.

The second prophylactic study was designed to optimize the potential effects IFNb based on the PK/PD studies described above. Given that interferon stimulated gene expression peaks between 2 and 4 h post administration, in this study all groups receiving IFNb treatments were started on treatment 2 h prior to infection and every other day thereafter. Moreover, to maximize the potential IFN effect in the IFNb only group, we utilized a 25× human equivalent dose rather than utilize a 1× human equivalent dose as done in the previous prophylactic study. In randomly assigned groups of 12–14-week-old Ces1c−/− hDPP4 female mice, we compared vehicle, LPV/RTV-IFNb low (1.6 MIU/kg or 4.8E4 IU/30 g mouse (40 µg/30 g mouse)), LPV/RTV-IFNb high (40 MIU/kg or 1.2 MIU/30 g mouse (1000 µg/30 g mouse)), and IFNb high alone. The LPV/RTV dose amounts and schedule were similar to that in the previous study. To control for dosing effects, vehicle-treated mice received both oral vehicle and subcutaneous PBS to mirror IFNb injections. Likewise, IFNb only group received oral vehicle to mirror that seen in orally dosed groups. Similar to the previous study, RDV and its vehicle were administered as a control. MERS-CoV infection was performed exactly as described in the previous study. Mice were weighed daily to monitor morbidity. Unlike previous studies, pulmonary function measurements by WBP (Data Sciences International) were performed on all mice in all groups until 2 dpi, after which these measurements were performed on all remaining mice per group (n = 6)28. On 2 dpi, three animals per group were killed by isofluorane overdose, lungs were scored for hemorrhage (described above), and the large left lobe was frozen at −80 °C for viral titration via plaque assay22. On 6 dpi, animals were killed and processed as done on 2 dpi.

Therapeutic in vivo efficacy studies

For head-to-head therapeutic efficacy studies comparing RDV to LPV/RTV-IFNb, female 9–12-week-old Ces1c−/− hDPP4 were randomly assigned to each treatment group (n = 10–12). After a 5–7 day acclimation time at BSL3, mice were anaesthetized with a mixture of ketamine/xylazine and then intranasally infected with 5E + 04 pfu of MERS M35C4 in 50 µl virus collection medium (see above). One day post infection, treatment was initiated. For LPV/RTV groups, mice were administered a human equivalent dose of a coformulation of LPV (160 mg/kg) and RTV (40 mg/kg) at 5 mL/kg once daily via oral gavage. Animals that received LPV/RTV also received mouse IFNb (R + D Systems) every other day at one of two doses via subcutaneous injection. The high IFNb dose group was administered a 25× human equivalent dose of 40 MIU/kg or 1.2 MIU/30 g mouse (1000 µg/30 g mouse). The low IFNb dose group was administered as 1× human equivalent dose of 1.6 MIU/kg or 4.8E4 IU/30 g mouse (40 µg/30 g mouse). To control for potential vehicle effects in the LPV/RTV-IFNb groups, we administered oral vehicle (propylene glycol, ethanol) daily and subcutaneously injected with PBS every other day. RDV (25 mg/kg) at 10 mL/kg was administered twice daily via subcutaneous injection. As a control for RDV, an additional group was given subcutaneous vehicle as a control. To monitor morbidity, mice were weighed daily. A subset of each cohort was randomly assigned for pulmonary function measurements by WBP (Data Sciences International) daily28. On 6 dpi, animals were killed by isoflurane overdose, lungs were scored for hemorrhage (described above), and the large left lobe was frozen at −80 °C for viral titration via plaque assay as described above22. The inferior right lobe was placed in 10% buffered formalin and stored at 4 °C for 1–3 weeks, until sectioning and histological analysis. Lung sectioning, hematoxylin and eosin staining, as well as MERS-CoV antigen staining as described above were performed by the Animal Histopathology & Laboratory Medicine Core at UNC.

Acute lung injury histological assessment tools

We used two different and complementary quantitative histologic tools to determine if antiviral treatments diminished the histopathologic features associated with lung injury. Both analyses and scoring were performed by a Board Certified Veterinary Pathologist who was blinded to the treatment groups.

The first tool is a Lung Injury Scoring System that was created by the American Thoracic Society in order to help quantitate histological features of ALI observed in mouse models and increase their translation to the human condition26. In a blinded manner, we chose three random diseased fields of lung tissue at high power (60 ×), which were scored for the following: (A) neutrophils in the alveolar space (none = 0, 1–5 cells = 1, > 5 cells = 2), (B) neutrophils in the interstitial space/septae (none = 0, 1–5 cells = 1, > 5 cells = 2), (C) hyaline membranes (none = 0, one membrane = 1, > 1 membrane = 2), (D) Proteinaceous debris in air spaces (none = 0, one instance = 1, > 1 instance = 2), (E) alveolar septal thickening (< 2× mock thickness = 0, 2–4× mock thickness = 1, > 4× mock thickness = 2). To obtain a lung injury score per field, the scores for A–E were then put into the following formula, which contains multipliers that assign varying levels of importance for each phenotype of the disease state.: score = [(20x A) + (14 x B) + (7 x C) + (7 x D) + (2 x E)]/100. The scores for the three fields per mouse were averaged to obtain a final score ranging from 0 to and including 1.

The second histological tool to quantitate lung injury was reported by Schmidt et al., where they used this tool to quantitate diffuse alveolar damage (DAD) in mice infected with RSV29. DAD is the pathological hallmark of ALI26,29,46. Similar to the implementation of the ATS tool described above, we scored three random diseased fields of lung tissue at high power (60 × ) for the following in a blinded manner: 1 = absence of cellular sloughing and necrosis, 2 = Uncommon solitary cell sloughing and necrosis (1–2 foci/field), 3 = multifocal (3 + foci) cellular sloughing and necrosis with uncommon septal wall hyalinization, or 4 = multifocal ( >75% of field) cellular sloughing and necrosis with common and/or prominent hyaline membranes. The scores for the three fields per mouse were averaged to get a final DAD score per mouse.

Cleaved caspase-3 antigen quantitation

The active and cleaved form of caspase-3 can be differentiated by immunohistochemistry. Lung tissue sections were stained for cleaved caspase-3 antigen (1:500, Cell Signaling #9664) by the Animal Histopathology & Laboratory Medicine Core at UNC. At the UNC Translational Pathology Laboratory (TPL) Core, stained slides were scanned on an Aperio ScanScope XT (Leica Biosystems) with a 20X power objective and a camera resolution of 0.4942 microns per pixel. Images were analyzed in Definiens Architect XD 2.7 with Tissue Studio version 4.4.2. Within each tissue section, total area was calculated. Single cells were detected initially based on hematoxylin staining of nuclei, and cell borders were interpolated in Tissue Studio. Each cell was then scored for cleaved caspase staining intensity based on a linear scale of Low, Medium, and High scale. The sum of the percent positive for high and medium caspase-3 staining was graphed for our studies. Tissue sections of diseased mouse intestine was used as a positive control.

Statistical analysis

All statistical data analyses were performed in Graphpad Prism 7. Statistical significance for each endpoint was determined with specific statistical tests. For each test, a P-value < 0.05 was considered significant. Specific tests to determine statistical significance are noted in each sure legend.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.