COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms¹. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal², most experimental infections provide insights into mild disease³. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies⁴, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Extended Data Fig. 1.. Clinical and pathological disease in SARS-CoV-2-infected K18-hACE2 mice.

Mice were infected intranasally with 10⁵ PFU SARS-CoV-2. a. Viral RNA detected by qPCR targeting viral N gene with normalization to HPRT for the indicated organs at 2, 4, and 6 dpi (10³, n=4; 10⁴, n=4; 10⁵, n=6, each organ was collected from an individual mouse). b. Lungs from uninfected (n=3), and infected (day 4 (n=4) and day 6 (n=3) p.i.) mice were immunostained to detect SARS-CoV-2 N protein. Bar = 701 μm. c. Infected lungs exhibited evidence of airway edema (asterisks, bottom middle panel), alveolar hyaline membranes, vascular thrombosis (upper inset and arrow, bottom right panel), dying cells with pyknotic to karyorrhectic nuclei, and proliferative alveolar epithelium (arrowhead and lower inset, bottom right panel, arrows) Bar = 701 and 70 μm (top and bottom, respectively), H&E stain. d. Summary of lung lesion scoring, as described in Methods (uninfected, n=3; day 4, n=4; day 6, n=3, each brain was collected from an individual mouse). Two sections of each lung from 3-4 mice per group were evaluated. These data are representative of 3 independent experiments. (a, d). Data are shown as mean±SEM.

Extended Data Fig. 2.. Histological analysis of extrapulmonary tissue.

Mice were sacrificed at days 0, 4 and 6 p.i. with 10⁵ PFU SARS-CoV-2 and tissues prepared for histological examination. Liver (a), spleen (b), kidney (c), small intestine (d), and colon (e) were studied. Pathological changes were minor and only observed in the liver. In the liver, all mice had some blood vessels filled with clear space or aggregates variably composed of erythrocytes / platelets (insets). Rare vessels had evidence of eosinophilic fibrillar material adherent along the vascular wall consistent with fibrin thrombi (a, arrows, inset, middle panel) adherent along the vascular wall. Bar = 110 (a,c-e) and 221 μm (b), H&E stain. Two sections of each organ from 3-4 mice per group were evaluated.

Extended Data Fig. 3.. Inflammatory mediators and immune effector cells in infected lungs.

a. Cytokine and chemokine transcripts were measured by qPCR following reverse transcription of RNA isolated from the lungs of K18-hACE2 mice infected with 10⁵ PFU SARS-CoV-2 (mock and 6 dpi, n=3; 2 and 4 dpi, n=4, each lung was collected from an individual mouse), one independent experiment. Statistical significance compared to results obtained at 0 dpi. Data were analyzed using 2-tailed Student’s t tests without adjustments. b. Gating strategy for identification of immune cells in lungs is shown. c. Representative FACS plot of IFNγ⁺TNF⁺ CD8 and CD4 T cells (as gated in panel b) after stimulation with indicated peptide pools in the lungs of 10⁵ PFU SARS-CoV-2 infected K18-hACE2 mice. d. Summary data are shown (n=3 mice/time point). Data are representative of two independent experiments e. Quantification of immune cells (as gated in panel b) in the lungs (n=3 for uninfected group; n=4 for 4 and 6 dpi, each lung was collected from an individual mouse). Data are representative of two independent experiments. f. Sera were collected from infected mice at the indicated time points and IC₅₀ values determined by neutralization of SARS-CoV-2 pseudoviruses expressing luciferase (n=4, days 0,2,4,6; n=7, day 13, 2 independent experiments). (a, d-f) Data are shown as mean±SEM.

Extended Data Fig. 4.. Brain and nasal cavity infection in SARS-CoV-2 infected K18-hACE2 mice.

a. Brains from uninfected and infected (10⁵ PFU SARS-CoV-2) mice were immunostained to detect SARS-CoV-2 N protein b, c. Multiple sites were characterized by cellular and karyorrhectic nuclear debris (b, arrows). Thrombi were detected, seen here in thalamus (c, arrow and inset). Bar = 17 μm. d. Ependyma (arrow) at day 6 p.i. had focal denudation and degeneration of ependymal cells (arrowheads) overlying focal region of cellular and karyorrhectic nuclei debris (asterisks). e. Nerve bundles (NB) subjacent to OE had evidence of punctate to linear immunostaining (brown, arrows). f. Sites of N protein localization at interface of OE and RE at day 2 p.i. (see Fig. 1d) had evidence of cell death and cellular debris (arrows). g. OE with sustentacular cell immunostaining for N protein. Bar = 1.3 mm (a), 65 μm (b,c,e-g) and 130 μm (d). Two sections of each tissue from 3-4 mice per group were evaluated.

Extended Data Fig. 5.. Preference indices for social scent discrimination assays.

Preferences indices were calculated as described in Methods for each mouse shown in Figure 2. The preference index was calculated as time spent with preferred or nonpreferred scent: (preferred - nonpreferred) / (preferred + non preferred). a. For male mice, preferred scent = female dander; non preferred = male dander; (uninfected: n=15 mice, other groups: n=10 mice). b. For female mice, preferred scent = novel scent; nonpreferred = familiar scent; (uninfected and infected alone groups: n=8 mice each; infected/CP-treated groups: n=7 mice). Data were analyzed for statistical significance using 2-tailed Mann-Whitney U-tests and shown as mean±SEM.

Extended Data Fig. 6.. Effects of Convalescent plasma delivered 6 hours before or 24 hours after infection.

a. N protein immunostaining in the lungs of control (healthy donor) plasma or convalescent plasma (CP)-treated mice at 2 and 5 days p.i. with 10⁵ PFU SARS-CoV-2. Bar = 800 μm (top) and 160 μm (bottom). Two sections of each lung from 3 mice per group evaluated. Representative images are shown. b. Percentage of initial weight (left panel), and survival (right panel) of K18-hACE2 mice receiving control plasma (n=2) or undiluted CP (n=4) at 24 hours after challenge with 10⁵ PFU SARS-CoV-2. Data are shown as mean±SEM and are representative of one independent experiment. ANOVA and 2-tailed Student’s t tests without adjustments (weights) and log-rank (Mantel-Cox) tests (survival) were used to analyze these data.

Fig. 1.. Clinical and pathological disease in SARS-CoV-2-infected K18-hACE2 mice.

a. Percentage of initial weight and survival of wild type (n=1 mouse) and K18-hACE2 mice infected with 10³ (n=3 mice), 10⁴ (n=10 mice), or 10⁵ (n=8 mice) PFU SARS-CoV-2/mouse (2 independent experiments). ANOVA and 2-tailed Student’s t tests without adjustments (weight change) and log-rank (Mantel-Cox) tests (survival) were used to analyze these data. Left panel, P<0.0001 10³ vs 10⁵ PFU inoculum; p=0.0842 for 10⁴ vs 10⁵ PFU inoculum. b. Infectious virus titers detected by plaque assay in different organs at 2, 4, and 6 dpi with 10⁵ PFU SARS-CoV-2 (day 2, 4 n=4 mice; day 6, n=8 mice (brain) and n=4 (other organs). 3 independent experiments). LOD=limit of detection. c-j. Nasal and sinus tissue were examined at days 2 (c-f) and 5 (g-j). N protein immunostaining (c,d,e,g,i,j) and H&E stain (f,h). (a,b) Data are shown as mean±SEM. c. N protein immunostaining in olfactory epithelium (OE). d. N protein immunostaining (brown) localized near interface of OE and respiratory epithelium (RE). Subjacent to epithelium, N protein was occasionally detected in endothelial lining of vessels (left arrow) and Bowman’s glands (right arrow) were occasionally detected. e-f. Maxillary sinus (MS) lining epithelium had extensive immunostaining for N protein (e, brown, arrows) with common sloughing and cellular debris (f, arrowheads). The lateral nasal glands (LNG) also had multifocal cellular and karyorrhectic debris (f, arrows). g-h. OE at day 5 with N protein immunostaining (g, arrows) localized near interface with respiratory epithelium. At these sites, cellular sloughing and loss of cellularity (h, arrows) were seen. i-j. Strong N protein immunostaining (arrows) was seen in tall OE cells with “classic” morphology of sustentacular cells along with immunostain expanding to adjacent cells. Bars = 128 (c,e), 63 (g,h), 31 (d,f), 42 (i), and 21 (j) μm, respectively. Two sections of each tissue from 3-4 mice per group were evaluated.

Fig. 2.. SARS-CoV-2 infection causes anosmia in K18-hACE2 mice.

Male and female mice were treated with PBS (uninfected) or 10⁵ PFU SARS-CoV-2 intranasally. a,d. Schematic showing social scent discrimination tests. b,e. Weights were recorded daily. c. Each male mouse was allowed 5 minutes in the cage with male and female scent (bedding from male or female cages) placed at two corners of the cage. The time that male mice spent sniffing male or female scent was recorded. Data were analyzed by 2-way ANOVA. f. Female mice were exposed to their own bedding (‘familiar’) or bedding from another cage (‘novel’). The time that female mice spent exploring each bedding was recorded. Data were analyzed by 2-way ANOVA. In some experiments, mice were pretreated with undiluted convalescent plasma (results on right of vertical line (c,f)). g-i. Buried food test (schematic shown in g). Food was buried under the bedding and each mouse was allowed 4 minutes in the cage to search for the food. h. The dotted line denotes the time limit of 4 minutes. Data were analyzed by 2-tailed Mann-Whitney U-tests. i. The percentage of mice that found the buried food within 4 minutes is shown. Data were analyzed by χ² Fisher’s exact test. In some experiments, mice were pretreated with undiluted convalescent plasma (denoted by blue bars in h,i). Male mice: uninfected: n=15 mice; infected: n=10 mice. Female mice: uninfected and infected: n=8 mice each; infected/CP-treated: n=7 mice. analyzed in 4 independent experiments. (b,c,e,f,h,i) Data are shown as mean±SEM. Preferences indices for the social scent discrimination assays are shown in Extended Data 5.

Fig. 3.. Effects of convalescent plasma (CP) on outcomes.

a. Percentage of initial weight (left panel) and survival (right panel) of K18-hACE2 mice receiving control serum (n=4 mice, black), undiluted (n=4 mice, blue), 1:3 dilution (n=4 mice, red) and 1:9 dilution (n=3 mice, purple) human CP at 24 hours prior to challenge with 10⁵ PFU SARS-CoV-2. Data are from two independent experiments. ANOVA and 2-tailed Student’s t tests without adjustments (weight change) and log-rank (Mantel-Cox) tests (survival) were used to analyze these data. b. Viral titers of CP-treated mice in the lungs (left panel) and brains (right panel) at 2 and 5 dpi. n=3 except for 2 dpi, 1:9 dilution (n=2) mice, 1 independent experiment. LOD=limit of detection. 2-tailed Student’s t tests without adjustments were used to analyze these data. * P=0.0455, control vs. undiluted CP; P=0.0443 control vs. 1:3 diluted CP. c. Scores of N protein immunostaining in CP-treated mice in the nasal cavity at 2 (upper panel) and 5 (lower panel) dpi. 0 – none; 1 - rare <1%; 2 - multifocal or localized <33% cells; 3 - multifocal, coalescing, 33-66%; 4 - extensive >67%. Two sections of each sinonasal cavity from three mice per group were evaluated. Data are shown as mean±SEM. LNG-lateral nasal gland, RE-respiratory epithelium, OE-olfactory epithelium, VMO-vomeronasal organ.