The SARS-CoV-2 B.1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global Coronavirus Disease 2019 (COVID-19) pandemic resulting in millions of deaths worldwide. The extensive morbidity and mortality associated with the COVID-19 pandemic made the development of SARS-CoV-2 vaccines, antibody-based countermeasures, and direct acting antiviral agents a global health priority. As part of the development process, several key animal models of SARS-CoV-2 infection and lung pathogenesis were developed in mice, hamsters, nonhuman primates (NHP) and other animals for rapid testing and evaluation 3 . Remarkably, several highly effective vaccines and antibody therapeutics targeting SARS-CoV-2 spike protein gained regulatory approval and were deployed with hundreds of millions of doses given worldwide (https://covid19.who.int). While these measures markedly reduced numbers of infections, hospitalizations, and deaths, their e cacy has been jeopardized by emergence of highly transmissible variant SARS-CoV-2 strains with mutations in the spike protein that could compromise protective immune responses and therapeutics.
We also used whole-body plethysmography to measure pulmonary function in infected Syrian hamsters We next performed microcomputed tomography (micro-CT) to assess for lung abnormalities in hamsters at 7 dpi. We used a previously de ned CT severity score (see Legend and Methods) to evaluate animals for nodules, ground glass opacities, and regions of lung consolidation 35 . Micro-CT analysis revealed lung abnormalities in all B.1.617.2-infected hamsters on 7 dpi that were consistent with commonly reported imaging features of COVID-19 pneumonia 37 . In comparison, analysis of B.1.1.529-infected hamsters on 7 dpi revealed patchy, ill-de ned ground glass opacity consistent with minimal to mild pneumonia. Accordingly, Syrian hamsters infected with B.1.617.2 had a high CT disease score (~12), whereas those infected with B.1.1.529 had a substantially lower disease score (~2) that was only slightly greater than mock-infected hamsters (Fig 2j-k).
Macroscopically, the lungs obtained from the B.1.617.2-infected hamsters showed congestion and/or hemorrhage, but this was absent in B.1.1.529-infected animals ( Fig. 3a). At the microscopic level, immune cell in ltration and in ammation were present in the peribronchial regions of the lungs at 3 dpi with B.1.617.2. Moreover, at 6 days post B.1.617.2 infection, extensive in ltration of neutrophils and lymphocytes in the alveolar space and walls was accompanied by focal pulmonary edema and alveolar hemorrhage (Fig 3b, inset), and regenerative changes in the bronchial epithelia became prominent (Fig  3b). In contrast, in B.1.1.529-infected hamsters, small foci of in ammation in the alveoli and peribronchial regions were observed only at 6 dpi (Fig 3b). A semi-quantitative histopathology severity score of viral pneumonia at 6 dpi showed a worse score after B.1.617.2 than B.1.1.529 infection (Fig 3c).
Consistent with these ndings, after B.1.617.2 infection, viral RNA was detected readily in the alveoli and bronchial epithelia in lung tissue sections at 3 and 6 dpi (Fig 3d). In comparison, after B.1.1.529 infection, fewer bronchial epithelial cells and alveoli were positive for viral RNA at either time point (Fig 3d). Collectively, these ndings suggest that the B.1.1.529 Omicron variant replicates less e ciently in the lungs of Syrian hamsters, which results in less severe pneumonia compared to the B.1.617.2 Delta variant.
Although hamsters are susceptible to SARS-CoV-2 infection without a requirement for host adaptation and show some similarities to that observed in COVID-19 patients, they develop self-limiting clinical and respiratory disease. Even though hamster ACE2 can serve as a receptor for SARS-CoV-2 spike protein, some of the contact residues in human ACE2 are not conserved 38 , which could diminish infectivity. Moreover, ACE2 expression levels on particular cells in the respiratory tract may differ slightly between hamsters and humans, which could impact infectivity and clinical outcome. To develop a more susceptible hamster model, members of the consortium (Y.K.) used transgenic hamsters (generated by Z.W.) expressing human ACE2 under the epithelial cytokeratin-18 promoter 39 . Whereas intranasal inoculation of 10 3 PFU of HP-095 D614G virus resulted in marked weight loss within the rst week (Fig 2l) and uniform mortality by 10 dpi (Fig 2m), less weight loss and death (P < 0.05) were observed after infection with 10 3 PFU of B.1.1.529. Consistent with these clinical data, 1,000 to 10,000-fold lower levels of infectious virus were measured in the lungs of hACE2 transgenic hamsters challenged with B.1.1.529 compared to the HP-095 D614G virus at 3 and 5 dpi (Fig 2n). Notably, and as seen in wild-type Syrian hamsters, smaller differences in infection were observed in the nasal turbinates. Thus, B.1.1.529 infection in the lung is attenuated in both wild-type and hACE2 transgenic hamsters.

Discussion
Our experiments suggest that compared to other SARS-CoV-2 isolates (e.g., B.1.351 or B.1.617.2), the B.1.1.529 Omicron variant infection is attenuated in laboratory mice and hamsters for causing infection and/or disease. While these results are consistent with the very preliminary clinical data in humans suggesting that B.1.1.529 causes a more transmissible yet possibly milder respiratory infection 40,41 , the basis for the attenuation in rodents remains unknown. One pre-print study suggests that B.1.1.529 replicates faster in the human bronchus and less in lung cells, which may explain its greater transmissibility and putative lower disease severity 42 ; although it remains unclear if these observations extend to rodents, we observed less infection of hamster bronchial cells in vivo with B.1.1.529 Omicron than B.1.617.2 Delta virus. We also measured lower viral burden in nasal washes and turbinates in 129 mice compared to other SARS-CoV-2 strains. The attenuation in mice was unexpected given that B.1.1.529 has multiple mutations in the RBD that are sites (K417, E484, Q493, Q498, and N501) associated with adaptation for mice [23][24][25] . Moreover, the attenuation in hamsters also was surprising, given that all prior SARS-CoV-2 variants have replicated relatively equivalently and to high levels in this animal 35,43,44 . However, our results showing attenuation of B.1.1.529 in hamsters are consistent with another preliminary report 45 . Whereas the more than 30 substitutions (mutations, deletions, and Page 11/27 insertions) in the B.1.1.529 could impact receptor engagement and cell entry, sequence changes in other structural, non-structural, and immune evasion proteins could affects replication, temperature sensitivity, cell-to-cell spread, cell and tissue tropism, dissemination, and induction of pro-in ammatory immune responses in a species-speci c manner. Thus, detailed genetic and functional studies are required to de ne the basis of virological and clinical attenuation of B.1.1.529 in mice and hamsters.
Although we observed clinical attenuation of B.1.1.529 in mice and hamsters, these animals likely will still have utility in evaluating vaccine, antibody, or small molecule inhibitors. All of the mice and hamsters tested, to varying degrees, showed evidence of viral replication and dissemination to the lower respiratory tract, which could be prevented or mitigated by prophylactic or therapeutic countermeasures. The most severe B.1.1.529 infection and disease was observed in hACE2 expressing mice and hamsters, which is consistent with results with other SARS-CoV-2 strains and variants 14,22,39,46  Hamster infection experiments. Five-to-six-week-old male hamsters were obtained from Charles River Laboratories, Envigo, or Japan SLC Inc. The K18-hACE2 transgenic hamster line was developed with a piggyBac-mediated transgenic approach, in which the K18-hACE2 cassette from the pK18-hACE2 plasmid 12 was transferred into a piggyBac vector, pmhyGENIE-3 54 , for pronuclear injection. hACE2 transgenic hamsters will be described in detail elsewhere 39  ] Lungs were collected 4 dpi and homogenized in 1.0 mL DMEM, clari ed by centrifugation (1,000 x g for 5 min) and stored at -80°C. Nasal washes were clari ed by centrifugation (2,000 x g for 10 min) and the supernatant was stored at -80°C. To quantify viral load in lung tissue homogenates and nasal washes, RNA was extracted from 100 µL samples using E.Z.N.A. ® Total RNA Kit I (Omega) and eluted with 50 µL of water. Four microliters RNA was used for real-time RT-qPCR to detect and quantify N gene of SARS-CoV-2 using TaqMan™ RNA-to-CT 1-Step Kit (Thermo Fisher Scienti c) as described 57 . [Y.K.] The virus titers in the nasal turbinates and lungs were determined by plaque assay on Vero E6 cells expressing human TMRPSS2 as previously published 58 . [R.J.W.] RNA was extracted from clari ed nasal washes using the Qiagen RNeasy extraction kit (Qiagen, Hilden Germany) following the manufacturer's instructions. Samples were puri ed on the included columns and eluted in 50 µL of nuclease free water. PCR was conducted using 4X TaqMan Fast Virus Master Mix (Thermo Fisher) and the HKU N-gene primer/probe set.
Plaque assay. Vero-TMPRSS2 or Vero-TMPRSS2-hACE2 cells were seeded at a density of 1×10 5 cells per well in 24-well tissue culture plates. The following day, medium was removed and replaced with 200 μL of material to be titrated diluted serially in DMEM supplemented with 2% FBS. One hour later, 1 mL of methylcellulose overlay was added. Plates were incubated for 72 h, then xed with 4% paraformaldehyde A CT severity score, adapted from a human scoring system, was used to grade the severity of the lung abnormalities 59 . Each lung lobe was analyzed for degree of involvement and scored from 0-4 depending on the severity: 0 (none, 0%), 1 (minimal, 1%-25%), 2 (mild, 26%-50%), 3 (moderate, 51%-75%), or 4 (severe, 76%-100%). Scores for the ve lung lobes were summed to obtain a total severity score of 0-20, re ecting the severity of abnormalities across the three infected groups. Images were anonymized and randomized; the scorer was blinded to the group allocation.
Pathology. Excised animal tissues were xed in 4% paraformaldehyde in PBS, and processed for para n embedding. The para n blocks were cut into 3-µm-thick sections and mounted on silane-coated glass slides. Sections were processed for in situ hybridization using an RNA scope 2.5 HD Red Detection kit (Advanced Cell Diagnostics, Newark, California) with antisense probe targeting the nucleocapsid gene of SARS-CoV-2 (Advanced Cell Diagnostics). Speci c antigen-antibody reactions were visualized by means of 3,3'-diaminobenzidine tetrahydrochloride staining using the Dako Envision system (Dako Cytomation).
Lung tissue sections also were scored based on pathological changes. Scores were determined based on the percentage of alveolar in ammation in a given area of a pulmonary section collected from each animal in each group by using the following scoring system: 0, no pathological change; 1, affected area (≤10%); 2, affected area (<50%, >10%); 3, affected area (≥50%); an additional point was added when pulmonary edema and/or alveolar hemorrhage was observed.
Data availability. All data supporting the ndings of this study are available within the paper, in the Source Data, and from the corresponding author upon request. There are no restrictions in obtaining access to primary data.
Code availability. No code was used in the course of the data acquisition or analysis.