Early pathogenesis profiles across SARS-CoV-2 variants in K18-hACE2 mice revealed differential triggers of lung damages

Abstract

The on-going COVID-19 pandemic has given rise to SARS-CoV-2 clades and variants with differing levels of symptoms and severity. To this end, we aim to systematically elucidate the changes in the pathogenesis as SARS-CoV-2 evolved from ancestral to the recent Omicron VOC, on their mechanisms (e.g. cytokine storm) resulting in tissue damage, using the established K18-hACE2 murine model. We reported that among the SARS-CoV-2 viruses tested, infection profiles were initially similar between viruses from early clades but started to differ greatly starting from VOC Delta, where the trend continues in Omicron. VOCs Delta and Omicron both accumulated a significant number of mutations, and when compared to VOCs Alpha, Beta, and earlier predecessors, showed reduced neurotropism and less apparent gene expression in cytokine storm associated pathways. They were shown to leverage on other pathways to cause tissue damage (or lack of in the case of Omicron). Our study highlighted the importance of elucidating the response profiles of individual SARS-CoV-2 iterations, as their propensity of severe infection via pathways like cytokine storm changes as more variant evolves. This will then affect the overall threat assessment of each variant as well as the use of immunomodulatory treatments as management of severe infections of each variant.

Keywords: K18-hACE2 mice model; SARS-CoV-2; cytokine storm; immune response; variants of concern.

Figure 1

Summarized methodology of characterization of eight viral iterations (ancestral to Omicron) of SARS-CoV-2. K18-hACE2 mice were infected the eight different SARS-CoV-2 viruses and divided into survival group and 4 dpi group (sacrificed 4 days post infection). The survival group were used for assessment of survival and physiological assessment. The 4 dpi group were sacrificed and have organs harvested for virologic, histologic and molecular profiling analysis (cytokine and chemokine gene expressions). Figure created with BioRender.com.

Figure 2

SARS-CoV-2 viruses infected K18-hACE2 transgenic mice. Physiological conditions and weight changes of infected mice group across different SARS-CoV-2 viruses (L, V, G, GR, GRY-α, GH-β, GK-δ, and GRA-o), and uninfected mice over 14 dpi. Mice were infected with approximately 10³ PFU of the respective SARS-2-CoV viruses through intranasal delivery. (A) Survival percentage of infected mice over 14 days, (B) Significance comparison of survival curves for SARS-CoV-2 infected mice. Comparison of significance between survival curves of various variant infected mice using log-rank (Mantel-Cox) test. Cut off for p-value using Kaplan-Meier analysis was adjusted to 0.002 for multiple comparison between various survival curves. (C) percentage changes in body weight, (D) changes in physiological condition quantified through scoring of clinical symptoms. Individual mouse was scored across 14 days from 1 (normal) to 5 (worst) for five conditions: coat appearance, level of consciousness, activity, eye condition, and respiration. To determine clinical score of the infected mice, average of the five physiological parameters for each infection group. All infection groups, n=6 with except of variant G, n=5, and mock infected mice, n=3. Error bars denotes range for each time point. (E, F) For viral load comparison after infection, mice were sacrificed at 4 dpi (L, V, G, GR, GRY-α, GH-β: n = 7; GK-δ: n = 5; GRA-o: n = 6) with lungs, brain, liver, and spleen tissues harvested for titration. SARS-CoV-2 virus titre in lung and brain tissue determined via plaque assays. Geometric mean of viral titre is shown. Statistical significance between clade L and variants determined with two-tailed Mann-Whitney U test. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3

Histopathological and immunohistochemical analyses of lung sections from K18-hACE2 mice following infection with SARS-CoV-2 at 4 dpi. (A) Histopathological scoring of lung damage by evaluating the extent and areas of pulmonary involvement based on 6 criteria: inflammatory cell infiltrates, hemorrhage, edema, degeneration of alveolar epithelial cells, parenchymal wall expansion and bronchiole epithelial cell damage, assigned respectively with a score of 0 – 3. The average of the 6 parameters from mice for each group was regarded as the final histopathological scoring. Mice following mock infection are representative of n=3 per group while mice infected with SARS-CoV-2 are representative of n=7 per group (n=5 for GK-δ, and n=6 for GRA-o). (B) Hematoxylin and eosin staining of lung sections from mock- or SARS-CoV-2-infected K18-hACE2 mice. H&E-stained lung sections demonstrated massive inflammatory cell infiltrates, hemorrhage and thickened interalveolar septa in the mice following SARS-CoV-2 inoculation. Hemorrhage was observed in the mock-infected mice could be due to a crushing artifact and damaged capillary walls during tissue handling at necropsy. 50x total magnification, scale bars = 50µm. (C) Percentage of SARS-CoV-2 nucleocapsid protein positivity in the FFPE lung tissues infected with L, GRY-α, GH-β, GK-δ and GRA-o. (D) Detection of SARS-CoV-2 nucleocapsid protein (brown staining) showed the virus was predominantly found in alveolar septa and pneumocytes, but not bronchiole regions. DAB chromogen and hematoxylin counterstain were used. Each image is representative of n=7 per group (L, V, G, GR, GRY-α, GH-β), n=5 (GK-δ), n=6 (GRA-o), n=3 (mock). 5x total magnification, scale bars = 2000µm.

Figure 4

Comparison of 84 cytokines regulation changes among SARS-CoV-2 viruses at 4 dpi. Differentially regulated cytokines across all SARS-CoV-2 virus tested. Color scale indicating level of averaged fold-regulation changes across the groups, * indicate significant difference in fold-regulation changes (>2-fold, p<0.05) between infected and mock infected mice.

Figure 5

Comparison of differentially expressed genes in selected pathways relevant to cytokine storm and tissue damage at 4 dpi. Differentially regulated cytokines across all groups in pathways related to (A) cytokine storm; (B) cellular response to TNF (C) neutrophil chemotaxis (D) MAPK cascade (E) negative regulation of inflammatory responses (F) positive regulation of IL-17 production (G) positive regulation of T-cell mediated cytotoxicity. Color scale indicate level of averaged fold-regulation changes across the groups, * indicate significant difference in fold-regulation changes (>2-fold, p<0.05) between infected and mock infected mice.