SARS-CoV-2 Omicron-B.1.1.529 Variant leads to less severe disease than Pango B and Delta variants strains in a mouse model of severe COVID-19

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The B.1.1.529 Omicron variant is rapidly emerging and has been designated a Variant of Concern (VOC). The variant is highly transmissible and partially or fully evades a spectrum of neutralising antibodies due to a high number of substitutions in the spike glycoprotein. A major question is the relative severity of disease caused by the Omicron variant compared with previous and currently circulating variants of SARS-CoV-2. To address this, a mouse model of infection that recapitulates severe disease in humans, K18-hACE2 mice, were infected with either a Pango B, Delta or Omicron variant of SARS-CoV-2 and their relative pathogenesis compared. In contrast to mice infected with Pango B and Delta variant viruses, those infected with the Omicron variant had less severe clinical signs (weight loss), showed recovery and had a lower virus load in both the lower and upper respiratory tract. This is also reflected by less extensive inflammatory processes in the lungs. Although T cell epitopes may be conserved, the antigenic diversity of Omicron from previous variants would suggest that a change in vaccine may be required to mitigate against the higher transmissibility and global disease burden. However, the lead time to develop such a response may be too late to mitigate the spread and effects of Omicron. These animal model data suggest the clinical consequences of infection with the Omicron variant may be less severe but the higher transmissibility could still place huge burden upon healthcare systems even if a lower proportion of infected patients are hospitalised.


Introduction
Since the emergence of SARS-CoV-2 in late 2019 several new variants of concern (VOC) have emerged and been associated with waves of infection across the globe.
Most recently, the Delta VOC is estimated to have been responsible for over 99% of infections worldwide. The B.1.1.529 Omicron variant 1 of SARS-CoV-2 that emerged recently in South Africa is causing considerable concern 2 . This variant appears to be highly transmissible and has many substitutions in the spike glycoprotein as well as elsewhere in the genome raising fears that the virus may escape from pre-existing immunity, whether acquired by vaccination or by prior infection 3 . Several mutations in the receptor binding domain and S2 region of the spike protein are predicted to impact transmissibility and affinity for the ACE-2 receptor 4 .
Infection of humans with SARS-CoV-2 results in a range of clinical courses, from asymptomatic to severe disease and subsequent death in at risk individuals but also a small proportion of otherwise healthy individuals across all age groups. Severe infection in humans is typified by cytokine storms 5,6 , pneumonia, renlal failure and tissue specific immunopathological processes 7,8 . A small number of patients have no overt respiratory symptoms at all.
Animal models of COVID-19 present critical tools to fill knowledge gaps for the disease in humans. Compatibility with a more extensive longitudinal deep tissue sampling strategy and a controlled nature of infection are key advantages 9 . Different animal species can be infected with wild-type SARS-CoV-2 to serve as models of COVID-19, these include mice, hamsters, ferrets 10 and rhesus macaques and cynomolgus macaques 11 . The K18-hACE2 transgenic (K18-hACE2) mouse, where hACE2 expression is driven by the epithelial cell cytokeratin-18 (K18) promoter, was developed to study SARS-CoV pathogenesis 12 . This mouse is now widely used as a model that mirrors many features of severe COVID-19 infection in humans to develop understanding of the mechanistic basis of lung disease and to test pharmacological interventions 13,14 .
With the apparent high transmissibility of the Omicron variant as well as the ability to evade pre-existing immunity, we sought to rapidly assess the relative pathogenicity against previous isolates of SARS-CoV-2. To do this, K18-hACE2 mice were infected with a Pango B lineage variant, Delta and Omicron variants of SARS-CoV-2 and their relative pathogenesis and viral loads compared.

Infection of hACE2 mice with Omicron variant leads to less severe clinical signs and recovery.
To assess the relative pathogenicity of the Omicron variant, the established K18-hACE2 mouse model of SARS-CoV-2 was utilised 12 Fig. 1.  Infection of these mice with SARS-CoV-2 provides a model that resembles severe infection in humans that had not been vaccinated or treated with currently available licenced therapeutics. The major measure of disease was weight loss followed by end of experiment pathology. Throat swabs were taken daily for RT-qPCR to determine and compare viral loads.

Mice infected with Omicron variant have lower viral load
To determine the viral load in animals infected with each variant, total RNA was Omicron-infected mice than in the other two groups (approx. 100 fold) (Fig. 3B).
Similary, viral loads in the lungs were significantly lower (approx. 100 fold) in Omicron versus Pango B and Delta variant-infected mice (Fig. 3C)..

Mice infected with Omicron variant exhibit less severe pneumonia
To determine the extent of inflammatory changes in the lungs, the left lobes were examined grossly, by histology and by immunohistology for the detection of SARS-CoV-2 nucleoprotein expression. In animals infected with the Pango B or Delta variants, the lung showed large patchy areas of dark red discoloration (Fig. 4).
Histologically, a mild multifocal to diffuse increase in interstitial cellularity was seen.
This was accompanied by partly extensive areas where alveoli contained a few desquamed alveolar macrophages/type II pneumocytes, occasional degenerate cells as well as neutrophils, lymphocytes and macrophages. There were also areas where alveoli exhibited activated type II pneumocytes and occasional syncytial cells. There was extensive viral antigen expression in type I and II pneumocytes in both unaltered alveoli and those involved in the inflammatory processes. In the latter, macrophages were also found to be positive (Fig. 4). In addition, mild to moderate patchy to circular periarterial lymphocyte and macrophage dominated mononuclear infiltration, mild arteritis and vascular endothelial cell activation was also observed (Fig. 4), as previously described [16][17][18] . The lungs of Omicron variant infected mice appeared grossly widely unaltered (Fig. 4). Similarly, the histological examination revealed widely unaltered parenchyma, with focal mild increase in interstitial cellularity, small, more loosely consolidated areas (Fig. 4) with activated type II pneumocytes, occasional syncytial cells and degenerate cells, some desquamed macrophages/type II pneumocytes in alveolar lumina, and some infiltrating macrophages and neutrophils.
A few small patchy leukocyte aggregates (macrophages, neutrophils, lymphocytes) were occasionally observed adjacent to small arteries. Viral antigen expression was detected in 5 of the 6 animals, in type I and II pneumocytes and in macrophages in focal inflammatory processes (Fig. 4). A few arteries exhibited a mild vasculitis and/or mild patchy to circular mononuclear perivascular infiltrates.

Virus infection
Animals were randomly assigned into three cohorts. For SARS-CoV-2 infection, mice were anaesthetized lightly with isoflurane and inoculated intra-nasally with 50 µl containing 10 3 PFU SARS-CoV-2 in PBS. They were sacrificed at variable time-points after infection by an overdose of pentabarbitone. Animals were dissected and tissues collected immediately for downstream processing.

RNA extraction and DNase treatment
The upper lobes of the right lung were homogenised in 1ml of TRIzol reagent (Thermofisher) using a Bead Ruptor 24 (Omni International) at 2 m/s for 30 s. The homogenates were clarified by centrifugation at 12,000xg for 5 min before full RNA extraction was carried out according to manufacturer's instructions. RNA was quantified and quality assessed using a Nanodrop (Thermofisher) before a total of 1 μg was DNase treated using the TURBO DNA-free™ Kit (Thermofisher) as per manufacturer's instructions.

qRT-PCR for viral load
Viral loads were quantified using the GoTaq® Probe 1-Step RT-qPCR System cDNA was generated using the SuperScript IV reverse transcriptase kit (Thermofisher) and PCR carried out using Q5® High-Fidelity 2X Master Mix (New England Biolabs) as per manufacturer's instructions. Both PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and serially diluted 10-fold from 10 10 to 10 4 copies/reaction to form the standard curve.

Histology and immunohistology
The left lungs were fixed in 10% buffered formalin for 48 h and stored in 70% ethanol until trimming for histological examination and routinely paraffin wax embedding.
Consecutive sections (3-5 µm) were prepared and routinely stained with hematoxylineosin (HE) or subjected to immunohistochemistry (IHC) for the detection of SARS-CoV-2 antigen as previously described 16 . IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200-402-A50) and the horseradish peroxidase (HRP) method. Briefly, sections were deparaffinized and rehydrated through graded alcohol. Antigen retrieval was achieved by 20 min incubation in citrate buffer (pH 6.0) at 98 °C in a pressure cooker. This was followed by incubation with the primary antibody (diluted 1:6,000 in dilution buffer; Dako) overnight at 4 ⁰C, a 10 min incubation at RT with peroxidase blocking buffer (Agilent) and a 30 min incubation at RT with Envision+System HRP Rabbit (Agilent).
The reaction was visualized with diaminobenzidin (DAB; Dako) for 10 min at RT. After counterstaining with hematoxylin for 2 s, sections were dehydrated and coverslipped.