K18-hACE2 mice develop respiratory disease resembling severe COVID-19

SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Taken together, this suggests that this mouse model can be useful for studies of pathogenesis and medical countermeasure development.

TCID50 or 10 5 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and 23 uniform lethality in the 10 5 TCID50 group. High levels of viral RNA shedding were observed from 24 the upper and lower respiratory tract and intermittent shedding was observed from the intestinal 25 tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with 26 high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial 27 pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, 28 were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in 29 macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory 30 cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral 31 cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory 32 response and observed pathology bears resemblance to . Taken together, this suggests 33 that this mouse model can be useful for studies of pathogenesis and medical countermeasure 34 development. 35 36 37 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101 Introduction 49 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in Hubai province in 50 mainland China in December 2019, and is the etiological agent of coronavirus disease (COVID)-51 19 (1). SARS-CoV-2 can cause asymptomatic to severe lower respiratory tract infections in 52 humans, with early clinical signs including fever, cough and dyspnea (2, 3). Progression to severe 53 disease may be marked by acute respiratory distress syndrome (ARDS), with pulmonary edema, 54 bilateral diffuse alveolar damage and hyaline membrane formation (4-6). Although primarily a 55 respiratory tract infection, extra-respiratory replication of SARS-CoV-2 has been observed in 56 kidney, heart, liver and brain in fatal cases (7-9). Several experimental animal models for SARS-57 CoV-2 infection have been developed, including hamsters (10) ferrets (11) and non-human primate 58 models (12-15). SARS-CoV-2 pathogenicity within these animal models ranges only from mild to 59 moderate (10-15). Additional small animal models that recapitulate more severe disease 60 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint 7 lymphocytes; alveolar septal thickening, and distinctive vascular system injury (Fig 3a-3c). At 7 129 DPI, mice developed pulmonary pathology consisting of multifocal interstitial pneumonia 130 characterized by type II pneumocyte hyperplasia, septal, alveolar and perivascular inflammation 131 comprised of lymphocytes, macrophages and neutrophils, variable amounts of alveolar fibrin and 132 edema, frequent syncytial cells and single cell necrosis. Terminal bronchioles were similarly 133 affected and in the most severely affected areas fibrin and necrosis occluded the lumen (Fig 3e-134 3g). Immunohistochemistry (IHC) demonstrated viral antigen in pneumocytes and macrophages 135 of tissues on both 3 and 7 DPI (Fig 3d-3h). 136 We evaluated the localized infiltration of innate and adaptive immune cell populations at 3 and 7 137 DPI, as compared to control animals and the survivor at 21 DPI. An absence of immunoreactive 138 macrophages (CD68+) in the γ-irradiated SARS-CoV-2 inoculated controls was noted (Fig 4a). In 139 contrast, in lung tissue of infected animals, an infiltration of a limited number of macrophages at 140 3 and 7 DPI was seen, which persisted in the survivor up until 21 DPI (Fig 4 d, g and j). We next 141 assessed lymphocyte infiltration into the lung in more detail. T cells were present in low numbers 142 in the non-infected control (Fig 4b). At 3 DPI T cells numbers increases in perivascular tissue and 143 alveolar septa and persisted through 7 DPI. B cells were present in low numbers in the γ-irradiated 144 SARS-CoV-2 inoculated controls and at 3 DPI, increased numbers were observed in alveolar septa. 145 B cells persisted through 7 DPI, when they started to cluster and form aggregates. At 21 DPI, T 146 cells were found throughout the whole lung section and formation of lymphoid aggregates with B 147 cells in perivascular tissues was observed in the survivor (Fig 4e, h and k). Interestingly, this 148 animal also still demonstrated mildly inflamed alveolar septa which were often accompanied by 149 foamy macrophages within affected alveoli (S2 Fig). 150 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101 8 Both SARS-CoV-2 inoculated groups showed only limited lesions in the nasal turbinates at 3 and 151 7 DPI (Fig 5a-5b). IHC showed multifocal SARS-CoV-2 antigen in ciliated respiratory epithelial 152 cells (Fig 5c-5d). 153 At 3 DPI all brains were histologically normal (Fig 6a-6b). However, 7 DPI brain tissues showed 154 lesions raging from minimal to moderate and included lymphocytic perivascular cuffing, gliosis, 155 meningitis, encephalitis and microthrombi, a generalized increase in cellularity of the meninges, 156 cerebral cortex and hippocampus and presence of edema (Fig 6c-6d). Abundant SARS-CoV-2 157 antigen was detected in the cerebral cortex and hippocampus within neurons and glial cells along 158 the soma and axons (Fig 6e-6f). In addition, cerebral cortex contained microthrombi and an 159 increased glial cell count, infiltration of inflammatory cells and scant hemorrhage (Fig 6g-6f). 160 161

Rapid humoral immune response in SARS-CoV-2-inoculated K18-hACE mice 162
We next investigated two key aspects of the anti-viral immune response. To assess B-cell response 163 and class-switch, the presence of SARS-CoV-2 spike-specific immunoglobulin (Ig)G and IgM 164 antibodies in serum obtained at 3 and 7 DPI was investigated using ELISA. By 3 DPI, one mouse 165 in the high dose group was positive for IgM and no mice were positive for IgG. In contrast, both 166 spike-specific IgM and IgG were found in sera of all mice at 7 DPI (Fig 7a). IgM and IgG titers of 167 one surviving animal at 21 DPI were comparable to those at 7 DPI. 168

2-inoculated K18-hACE mice 171
To investigate the immune response further we utilized serum multiplex cytokine analysis to 172 characterize the inflammatory status and identify key patterns. Interestingly, while serum cytokine 173 9 levels at 3 DPI showed only slight changes as compared to control animals, strong upregulation 174 was observed for multiple cytokines and chemokines by 7 DPI (Fig 7b). A strong increase in T 175 helper (Th)1-mediated cytokines interferon (IFN)-g (both doses, p = 0.0268, 0.0268) and tumour 176 necrosis factor (TNF)-a, (though not statistically significant) was observed. In addition, there was 177 also an upregulation of proinflammatory and chemoattractant cytokine IFN-g-induced protein (IP)-178 10 (C-X-C motif chemokine ligand (CXCL10)) (high dose, p = 0.0268). Interestingly, no trend of 179 upregulation of Th2 anti-inflammatory cytokines interleukin (IL)-4 and IL-5 was seen, but 180 increased levels of IL-10 were observed at DPI 7 in both groups, which has been shown to have an MCP-1 (35-37). In the lungs of aged hACE2 mice, SARS-CoV-2 infection leads to elevated 220 cytokine production including Eotaxin, G-CSF, IFN-γ, IL-9, and MIP-1β (38). Here, we show that 221 SARS-CoV-2 infection of K18-hACE2 mice elicits a measurable systemic pro-inflammatory 222 cytokine response which is significantly increased at 7 DPI and characterized by an increase in 223 IFN-g, TNF-a and IP-10, and also encompasses upregulation of innate cell-recruiting chemokines 224 GM-CSF and MCP-1. Importantly, increased levels of IFN-γ, IP-10, MCP-1 and TNF-a are 225 associated with severity of disease in in 39,40). COVID-19 patients also 226 show heightened IL-4 and IL-10 levels, cytokines associated with inhibitory inflammatory 227 responses (41). While the K18-hACE2 model did not recapitulate IL-4 upregulation, increased IL-228 10 levels were observed in serum, suggesting that both pro-and anti-inflammatory cytokine 229 response are functioning in this mouse model. This is particularly relevant, as in COVID-19, the 230 resulting cytokine storm is not only thought to be detrimental to disease progression but also 231 closely linked to the development of ARDS (39). In addition, cytokine levels are also reported to 232 be indicative of extrapulmonary multiple-organ failure (42,43). Reports suggest that upregulation 233 of IL-6, IL-8, and TNF-α contributes to SARS-related ARDS (35,44). Interestingly, while we did 234 observe the upregulation of TNF-α, IL-6 levels remained unchanged. This needs to be further 235 investigated to clarify if our observation suggests a differently modulated immune response and 236 pathogenesis that should be considered for intervention studies. 237 We have also demonstrated a functional humoral immune response and production of both IgM 238 and IgG antibodies. This is in line with observations made in ACE2-HB-01 mice where IgG 239 antibodies against spike protein of SARS-CoV-2 were also observed (26). This indicates that the 240 K18-hACE2 mouse model mounts a robust innate and adaptive immune response. 241 12 The mouse model presented here recapitulates histopathological findings of COVID-19 associated 242 ARDS, a robust innate and adaptive immune-response, neurological involvement and, importantly, 243 presents a dose-dependent sub-lethal disease manifestation. As such, we believe this model to be 244 highly suitable for testing of SARS-CoV-2 countermeasures such as antiviral and immune-245 modulatory interventions. However, COVID-19 associated ARDS in patients presents not just 246 with characteristic lung pathology, but also with clinical manifestations including hypoxia, loss of 247 lung compliance and requirement for intubation, liver and kidney involvement and associated 248 increase in serum protein levels, and decreased lymphocyte numbers. To accurately assess how 249 well K18-hACE2 mice recapitulates human ARDS, additional studies specifically addressing 250 these aspects are required. Institutional Biosafety Committee (IBC) approved work with infectious SARS-CoV-2 virus strains 261 under BSL3 conditions. All sample inactivation was performed according to IBC approved 262 standard operating procedures for removal of specimens from high containment. 263 264 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint Samples were collected with prewetted swabs in 1 mL of DMEM supplemented with 100 U/mL 285 penicillin and 100 μg/mL streptomycin. Then, 140 µL was utilized for RNA extraction using the 286 QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the 287 105 and is also made available for use under a CC0 license.

Cells and virus
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint manufacturer's instructions with an elution volume of 150 µL. Tissues (up to 30 mg) were 288 homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to 289 the manufacturer's instructions. Viral RNA was detected by qRT-PCR (46). Five μL RNA was 290 tested with the Rotor-GeneTM probe kit (Qiagen) according to instructions of the manufacturer. 291 Ten-fold dilutions of SARS-CoV-2 standards with known copy numbers were used to construct a 292 standard curve. instructions. Samples were pre-diluted 1:3 in the kit serum matrix (v:v). Concentrations below the 310 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint limit of detections were set to zero. Heatmap and correlation graphs were made in R (47) using 311 pheatmap (48) and corrplot (49)

Statistical analyses 332
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101 Two-tailed Mann-Whitney's rank tests and Wilcoxon matched-pairs rank test were conducted to 333 compare differences between groups.

362
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint 373 and other lineage B betacoronaviruses. Nature microbiology. 2020;5(4):562-9.

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(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020.

437
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint  105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 11, 2020. . https://doi.org/10.1101/2020.08.11.246314 doi: bioRxiv preprint