Roborovski dwarf hamster model for the assessment of antiviral protection

For in vivo experiments, SARS-CoV-2 isolates BetaCoV/Germany/BavPat1/2020 and BetaCoV/Germany/ChVir21652/2020 (B.1.1.7) were grown on Vero E6 cells and whole-genome sequenced before infection experiments to confirm genetic integrity. All virus stocks were titrated on Vero E6 cells before infection.

A total of 120 female (67) and male (53) Roborovski dwarf hamsters (Phodopus roborovskii) obtained via the German pet trade were used for infection experiments. Animals were housed in groups of 3–6 animals of the same sex in individually ventilated GR900 cages (Tecniplast) and provided with food and water ad libitum and bountiful enrichment (Carfil). Infection was performed by intranasal administration of 1 × 10⁵ PFU of SARS-CoV-2 in 20 µl of cell culture medium under general anesthesia³⁶. All animal procedures were performed in accordance with relevant institutional and legal regulations and approved by the responsible state authority: Landesamt für Gesundheit und Soziales, Berlin, Germany, permit number G 0086/20.

DARPin molecules and mAbs were administered intraperitoneally in sterile PBS. The final drug concentration was adjusted based on the desired dose and respective animal weight to a 100-µl injection volume. All animals in this study were treated once at the indicated timepoint, 0 hours, 6 hours or 24 hours p.i.

From a total of 120 Roborovski dwarf hamsters, 54 were used to determine dose and time dependency of treatment success. Six animals (four female and two male) per group were infected with 1 × 10⁵ PFU of SARS-CoV-2 wild-type (BetaCoV/Germany/BavPat1/2020) and treated with 3 mg kg⁻¹, 10 mg kg⁻¹ or 20 mg kg⁻¹ of ensovibep at the time of infection, with 1 mg kg⁻¹ or 20 mg kg⁻¹ 6 hours p.i. or with 10 mg kg⁻¹ 24 hours p.i. A placebo (PBS) treatment group with six animals (four female and two male) was also included in each of three studies performed for this purpose. Results of these experiments are summarized in Supplementary Fig. 11.

To compare efficacy of ensovibep and mAb cocktail treatment, 60 animals were infected with 1 × 10⁵ PFU of SARS-CoV-2 variant B.1.1.7 (BetaCoV/Germany/ChVir21652/2020). Subjects were divided into groups of 12 animals (six female and six male) and treated with 10 mg kg⁻¹ of ensovibep, 10 mg kg⁻¹ of Regeneron mAb cocktail or placebo (PBS) at the time of infection or with 10 mg kg⁻¹ of ensovibep or 10 mg kg⁻¹ of Regeneron mAb cocktail 24 hours p.i. An additional six (three female and three male) animals served as a non-infected control group. Results of this experiment are presented in Figs. ​Figs.44–6.

In all in vivo infection experiments performed in this study, half of each respective group was scheduled for take-out at 3 days p.i.; the other half was to be terminated at 5 days p.i. In some of the experiments, several animals had to be terminated at timepoints other than these for humane reasons. Defined humane endpoints included body temperature <33 °C, body weight loss >15% together with signs of respiratory distress, body weight loss >20% or a combination of these factors. Animals were monitored at least twice a day to prevent any prolonged suffering.

RNA was extracted from throat swabs and lung tissue using the innuPREP Virus DNA/RNA Kit (Analytic Jena). Viral RNA was quantified using a one-step RT–qPCR reaction with the NEB Luna Universal Probe One-Step RT–qPCR (New England Biolabs) and the 2019-nCoV RT–qPCR primers and probe (E_Sarbeco)⁷³ on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), as previously described³⁶. To obtain virus titers, duplicate ten-fold serial dilutions of lung tissue homogenates were made and incubated on Vero E6 monolayers for 2 hours at 37 °C. Cells were washed and overlaid with semi-solid cell culture medium containing 1.5% microcrystalline cellulose (Avicel) and incubated for 48 hours at 37 °C. Plates were then fixed with 4% formalin and stained with 0.75% crystal violet for plaque counting.

For histopathology, the left lung lobe was carefully removed, immersion-fixed in formalin, pH 7.0, for 48 hours, embedded in paraffin and cut in 2-μm sections. Slides were stained with hematoxylin and eosin (H&E) after de-waxing in xylene and rehydration in decreasing ethanol concentrations. Lung sections were microscopically evaluated in a blinded fashion by a board-certified veterinary pathologist to assess the character, distribution and severity of pathologic lesions using lung-specific inflammation scoring parameters as previously described for other lung infection models. Three different scores were used that included the following parameters: (1) lung inflammation score including severity of (i) interstitial pneumonia, (ii) bronchiolitis, (iii) necrosis of bronchial and alveolar epithelial cells and (iv) hyperplasia of alveolar epithelial type II cells as well as (v) hyperplasia of bronchial epithelial cells; (2) immune cell infiltration score taking into account the presence of (i) neutrophils, (ii) macrophages and (iii) lymphocytes in the lungs as well as (iv) perivascular lymphocytic cuffing; and (3) edema score including (i) alveolar edema and (ii) perivascular edema. H&E-stained slides were analyzed, and images were taken using an Olympus BX41 microscope with a DP80 Microscope Digital Camera and cellSens Imaging Software, version 1.18 (Olympus). For the display of overviews of whole lung lobe sections, slides were automatically digitized using the Aperio CS2 slide scanner (Leica Biosystems Imaging), and image files were generated using Image Scope Software (Leica Biosystems Imaging). The percentages of lung tissues affected by inflammation were determined histologically by an experienced board-certified experimental veterinary pathologist (O.K.), as described previously⁷⁴. Lung inflammation scores were determined as absent, (1) mild, (2) moderate or (3) severe and quantified as described previously⁷⁴. Immune cell influx scores and edema scores were rated from absent to (1) mild, (2) moderate or (3) severe.