SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates

Abstract

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-β signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

Keywords: COVID-19; SARS-CoV-2; cytokines; inflammation; mouse adapted.

Fig. 1.

Passage of N501Y VIC2089 in lungs of adult C57BL/6 results in adaptive virulence mutations without affecting response to rechallenge. (A) Graphical representation of the serial passage method to generate SARS-CoV-2 P21 virus. The VIC2089 SARS-CoV-2 N501Y clinical isolate was used to infect C57BL/6 mice (n = 3). Animals were euthanized at 3 dpi, and lungs were homogenized, pooled, and used to infect a new cohort of animals (n = 3) intranasally. This process was repeated 21 times (the image was created with BioRender.com). (B and C) Mice were infected with 10⁴ TCID50 of passage 2, 5, 9, 14, or 21 (n = 8 to 10) and monitored at 3 dpi for (B) percent weight change compared to initial weight and (C) lung viral burden measured by TCID50 assay. (D and E) Mice were challenged intranasally with 10⁴ TCID50 of either the SARS-CoV-2 early passage P2 strain or the mouse adapted, P21 strain (n = 10 to 11) (D) Animals were euthanized at defined time points after infection, and lungs were collected for viral quantification by the TCID50 assay. (E) Daily percent weight change of infected animals compared to initial weight. (F) Virus passages were sequenced using next-generation sequencing, and strains showing nonsynonymous mutations were compared to the original clinical isolate (P2). In black (Top), a schematic of the SARS-CoV-2 mRNA is shown. P2, P5, P9, P14, and P21 mRNA representations are depicted with the acquired mutations marked on their respective locations on the mRNA. (G and H) Mock, P2-, or P21-infected mice were rechallenged 28 d later with P2 or P21 and analyzed 3 d after rechallenge for (G) percent weight change, compared to weight before second infection and (H) lung viral load (TCID50). Data were pooled from (B–E) or are representative of (G and H) 2 to 3 independent experiments. One-way ANOVA with multiple comparisons (B and C), 2-way ANOVA (D and E), and unpaired two-tailed Student’s t test after log₁₀ transformation (H) were performed. Mean ± SD is shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

P21 infection induces severe lung disease, cytokine storm, and changes in the host transcriptome. (A) Representative images of hematoxylin and eosin (H&E) and SARS-CoV-2 nucleocapsid stained lungs. Mice were infected intranasally with 10⁴ TCID50 of P2, P21, or mock (media only), and lungs were collected and fixed for histological analysis at days 2, 4, and 7 postinfection. Histological images are representative of at least 3 animals. Black arrows point to exemplary SARS-CoV-2-positive cells. (Scale bars, 50 µm.) (B) Mice were infected intranasally with 10⁴ TCID50 of either P2, P21, or inoculated with vehicle control (M = mock infection). Supernatants of lung homogenates were taken at days 2, 3, 4, 7, 14, and 28 postinfection for analysis of 26 cytokines/chemokines. The concentration of all measured cytokines/chemokines for each animal were summed, and mean ± SD of each cytokine is shown. Colours represent different analytes (n = 4 to 5, 6 to 8-wk old mice per group). (C) Cytokine analysis of infected mice over time. Lungs of animals infected with 10⁴ TCID50 of P2 or P21 were collected at days 2, 4, 7, 14, and 28 postinfection and utilized for ELISA of 26 different cytokines and chemokines. Each panel displays mock- (gray), P2- (blue), and P21- (red) infected animals. Boxplots of mock-infected samples depict the median and interquartile ranges. Loess smoothing was applied to the P2 and P21 infection time course, with the shaded area indicating 95% CIs. (D and E) Pathway enrichment analyses of significantly differentially expressed genes identified from P21- vs. mock-infected mice and P21- vs. P2-infected mice comparisons using Hallmark gene sets. Negative log₁₀ FDR-adjusted P values associated with each pathway are plotted; dot sizes correspond to the proportion of all genes from that pathway that were found to be significantly differentially expressed in a given comparison (Gene Ratio). Two-way ANOVA with multiple comparisons (B) and Wilcoxon rank-sum (C) statistical tests were performed; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.

Inflammation induced by SARS-CoV-2 variants is more pronounced in aged mice, and disease is driven by TNF, TGF-β, and E2F pathways. (A and B) C57BL/6 mice of different ages (young = 6 to 8 wk and aged = 6 to 8 mo) were intranasally inoculated with 10⁴ TCID50 SARS-CoV-2 P2 or P21 and monitored for (A) the proportion of mice that became moribund, reaching humane endpoint and (B) daily percentual weight change over time, relative to the initial weight. (n= 5 to 9 mice per group; results are representative of at least 3 independent experiments) (C) Representative images of hematoxylin and eosin (H&E), SARS-CoV-2 nucleocapsid, MPO (neutrophils), F4/80 (macrophages), or CD3 (T cells) stains of mouse lungs infected with P21 and harvested 3 dpi. Histological images are representative of at least 3 animals. (Scale bars, 50 µm.) (D) Average of sum of 26 cytokines/chemokines expressed in supernatants of lung homogenates of young and aged mice challenged intranasally with 10⁴ TCID50 of P2, P21, or mock at 3 dpi (n = 5 mice per group; mean ± SD of each cytokine is shown; one-way ANOVA with multiple comparisons was performed). (E) Levels of cytokine and chemokines in lung homogenates 3 dpi. Protein levels were measured by ELISA, and corresponding gene expression levels were quantified by bulk RNA-seq. Gene expression data are annotated by protein name rather than the original mouse gene identifier for each of comparison. The Wilcoxon rank-sum test, with Bonferroni adjustment for multiple comparisons, was performed between aged and young mice for each infection group. Boxplots depict the median and interquartile ranges. (F) Pathway enrichment analysis of significantly DE genes identified by directly comparing P21-infected aged and young animals at 3 dpi, using Hallmark gene sets. Negative log₁₀ false discovery rate–adjusted P values associated with each pathway are plotted; dot sizes correspond to the proportion of all genes from that pathway that were found to be significantly DE for a given comparison (Gene Ratio). (G) DE genes attributable to P21 infection in aged and young mice were converted to human homologs and performed a comparative pathway analysis between the two age groups using IPA. Among the top significantly enriched pathways for both age groups was “Role of Hypercytokinemia/Hyperchemokinemia in the Pathogenesis of Influenza” and “Coronavirus Pathogenesis”. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are representative of 2 to 3 independent experiments (A–C).

Fig. 4.

Severe pathology upon SARS-CoV-2 infection is less pronounced in animals lacking the gene encoding the proinflammatory cytokine TNF (A–C) Aged C57BL/6 and Tnf ^−/− mice (6 to 8 mo) were intranasally inoculated with 10⁴ TCID50 SARS-CoV-2 P21 and monitored for (A) the proportion of mice that became moribund and reached humane endpoint, (B) percent weight change 3 dpi, relative to the initial weight (n = 15 to 20 mice per group), and (C) lung viral loads as measured by TCID50 at 3 dpi (n = 4 to 7 mice per group). (D–F) C57BL/6 and Tnf ^−/− mice (6 to 8-wk-old) were intranasally inoculated with 10⁴ TCID50 SARS-CoV-2 P21 strain and monitored for (D) percent weight change, relative to the initial weight, (E) lung TCID50 at day 3 postinfection [n = 23 to 24 mice per group (D and E)], and (F) average of sum of 26 cytokines/chemokines expressed in supernatants of lung homogenates (n = 5 mice per group; mean ± SD of each cytokine is shown; mock WT mice were challenged with vehicle only). (G and H) Adult C57BL/6 (10 to 12 wk) were treated with etanercept (anti-TNF) or vehicle and monitored for the proportion of mice that reached humane endpoint (>20% weight loss) after intranasal inoculation with 10⁴ TCID50 SARS-CoV-2 P21. Treatment was commenced either (G) at the time of infection (n = 8 to 9 mice per group) or (H) 2 wk prior to infection (n = 18 mice per group, pooled from 3 independent experiments). Mean ± SD is shown (B–F). Log-rank Mantel–Cox test (A, G, and H), one-way ANOVA (F), and unpaired two-tailed Student’s t test (B and D) after log₁₀ transformation (C and E) were performed; *P < 0.05 and ****P < 0.0001.