Global analysis of lysine acetylation in the brain cortex of K18-hACE2 mice infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected hundreds of millions of people all over the world and thus threatens human life. Clinical evidence shows that SARS-CoV-2 infection can cause several neurological consequences, but the existing antiviral drugs and vaccines have failed to stop its spread. Therefore, an understanding of the response to SARS-CoV-2 infection of hosts is vital to find a resultful therapy. Here, we employed a K18-hACE2 mouse infection model and LC-MS/MS to systematically evaluate the acetylomes of brain cortexes in the presence and absence of SARS-CoV-2 infection. Using a label-free strategy, 3829 lysine acetylation (Kac) sites in 1735 histone and nonhistone proteins were identified. Bioinformatics analyses indicated that SARS-CoV-2 infection might lead to neurological consequences via acetylation or deacetylation of important proteins. According to a previous study, we found 26 SARS-CoV-2 proteins interacted with 61 differentially expressed acetylated proteins with high confidence and identified one acetylated SARS-CoV-2 protein nucleocapsid phosphoprotein. We greatly expanded the known set of acetylated proteins and provide the first report of the brain cortex acetylome in this model and thus a theoretical basis for future research on the pathological mechanisms and therapies of neurological consequences after SARS-CoV-2 infection.

De Novo Human Angiotensin-Converting Enzyme 2 Decoy NL-CVX1 Protects Mice From Severe Disease After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections.

Aerosolized sulfated hyaluronan derivatives prolong the survival of K18 ACE2 mice infected with a lethal dose of SARS-CoV-2.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

SARS-CoV-2 Omicron variant causes brain infection with lymphoid depletion in a mouse COVID-19 model.

BACKGROUND: The Omicron variant has become the most prevalent SARS-CoV-2 variant. Omicron is known to induce milder lesions compared to the original Wuhan strain. Fatal infection of the Wuhan strain into the brain has been well documented in COVID-19 mouse models and human COVID-19 cases, but apparent infections into the brain by Omicron have not been reported in human adult cases or animal models. In this study, we investigated whether Omicron could spread to the brain using K18-hACE2 mice susceptible to SARS-CoV-2 infection. RESULTS: K18-hACE2 mice were intranasally infected with 1 × 105 PFU of the original Wuhan strain and the Omicron variant of SARS-CoV-2. A follow-up was conducted 7 days post infection. All Wuhan-infected mice showed > 20% body weight loss, defined as the lethal condition, whereas two out of five Omicron-infected mice (40%) lost > 20% body weight. Histopathological analysis based on H&E staining revealed inflammatory responses in the brains of these two Omicron-infected mice. Immunostaining analysis of viral nucleocapsid protein revealed severe infection of neuron cells in the brains of these two Omicron-infected mice. Lymphoid depletion and apoptosis were observed in the spleen of Omicron-infected mice with brain infection. CONCLUSION: Lethal conditions, such as severe body weight loss and encephalopathy, can occur in Omicron-infected K18-hACE2 mice. Our study reports, for the first time, that Omicron can induce brain infection with lymphoid depletion in the mouse COVID-19 model.

Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has been a global health concern since 2019. The viral spike protein infects the host by binding to angiotensin-converting enzyme 2 (ACE2) expressed on the cell surface, which is then processed by type II transmembrane serine protease. However, ACE2 does not react to SARS-CoV-2 in inbred wild-type mice, which poses a challenge for preclinical research with animal models, necessitating a human ACE2 (hACE2)-expressing transgenic mouse model. Cytokeratin 18 (K18) promoter-derived hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] are widely used for research on SARS-CoV-1, MERS-CoV, and SARS-CoV-2. However, SARS-CoV-2 infection is lethal at ≥105 PFU and SARS-CoV-2 target cells are limited to type-1 alveolar pneumocytes in K18-hACE2 mice, making this model incompatible with infections in the human lung. Hence, we developed lung-specific SARS-CoV-2 infection mouse models with surfactant protein B (SFTPB) and secretoglobin family 1a member 1 (Scgb1a1) promoters. After inoculation of 105 PFU of SARS-CoV-2 to the K18-hACE2, SFTPB-hACE2, and SCGB1A1-hACE2 models, the peak viral titer was detected at 2 days post-infection and then gradually decreased. In K18-hACE2 mice, the body temperature decreased by approximately 10°C, body weight decreased by over 20%, and the survival rate was reduced. However, SFTPB-hACE2 and SCGB1A1-hACE2 mice showed minimal clinical signs after infection. The virus targeted type I pneumocytes in K18-hACE2 mice; type II pneumocytes in SFTPB-hACE2 mice; and club, goblet, and ciliated cells in SCGB1A1-hACE2 mice. A time-dependent increase in severe lung lesions was detected in K18-hACE2 mice, whereas mild lesions developed in SFTPB-hACE2 and SCGB1A1-hACE2 mice. Spleen, small intestine, and brain lesions developed in K18-hACE2 mice but not in SFTPB-hACE2 and SCGB1A1-hACE2 mice. These newly developed SFTPB-hACE2 and SCGB1A1-hACE2 mice should prove useful to expand research on hACE2-mediated respiratory viruses.

Upper Respiratory Infection Drives Clinical Signs and Inflammatory Responses Following Heterologous Challenge of SARS-CoV-2 Variants of Concern in K18 Mice.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of several variants of concern (VOC) with increased immune evasion and transmissibility. This has motivated studies to assess protection conferred by earlier strains following infection or vaccination to each new VOC. We hypothesized that while NAbs play a major role in protection against infection and disease, a heterologous reinfection or challenge may gain a foothold in the upper respiratory tract (URT) and result in a self-limited viral infection accompanied by an inflammatory response. To test this hypothesis, we infected K18-hACE2 mice with SARS-CoV-2 USA-WA1/2020 (WA1) and, after 24 days, challenged with WA1, Alpha, or Delta. While NAb titers against each virus were similar across all cohorts prior to challenge, the mice challenged with Alpha and Delta showed weight loss and upregulation of proinflammatory cytokines in the URT and lower RT (LRT). Mice challenged with WA1 showed complete protection. We noted increased levels of viral RNA transcripts only in the URT of mice challenged with Alpha and Delta. In conclusion, our results suggested self-limiting breakthrough infections of Alpha or Delta in the URT, which correlated with clinical signs and a significant inflammatory response in mice.

Virulence Profiles of Wild-Type, P.1 and Delta SARS-CoV-2 Variants in K18-hACE2 Transgenic Mice.

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice.

Proteomic and phosphoproteomic characteristics of the cortex, hippocampus, thalamus, lung, and kidney in COVID-19-infected female K18-hACE2 mice.

BACKGROUND: Neurological damage caused by coronavirus disease 2019 (COVID-19) has attracted increasing attention. Recently, through autopsies of patients with COVID-19, the direct identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in their central nervous system (CNS) has been reported, indicating that SARS-CoV-2 might directly attack the CNS. The need to prevent COVID-19-induced severe injuries and potential sequelae is urgent, requiring the elucidation of large-scale molecular mechanisms in vivo. METHODS: In this study, we performed liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analyses of the cortex, hippocampus, thalamus, lungs, and kidneys of SARS-CoV-2-infected K18-hACE2 female mice. We then performed comprehensive bioinformatic analyses, including differential analyses, functional enrichment, and kinase prediction, to identify key molecules involved in COVID-19. FINDINGS: We found that the cortex had higher viral loads than did the lungs, and the kidneys did not have SARS-COV-2. After SARS-CoV-2 infection, RIG-I-associated virus recognition, antigen processing and presentation, and complement and coagulation cascades were activated to different degrees in all five organs, especially the lungs. The infected cortex exhibited disorders of multiple organelles and biological processes, including dysregulated spliceosome, ribosome, peroxisome, proteasome, endosome, and mitochondrial oxidative respiratory chain. The hippocampus and thalamus had fewer disorders than did the cortex; however, hyperphosphorylation of Mapt/Tau, which may contribute to neurodegenerative diseases, such as Alzheimer's disease, was found in all three brain regions. Moreover, SARS-CoV-2-induced elevation of human angiotensin-converting enzyme 2 (hACE2) was observed in the lungs and kidneys, but not in the three brain regions. Although the virus was not detected, the kidneys expressed high levels of hACE2 and exhibited obvious functional dysregulation after infection. This indicates that SARS-CoV-2 can cause tissue infections or damage via complicated routes. Thus, the treatment of COVID-19 requires a multipronged approach. INTERPRETATION: This study provides observations and in vivo datasets for COVID-19-associated proteomic and phosphoproteomic alterations in multiple organs, especially cerebral tissues, of K18-hACE2 mice. In mature drug databases, the differentially expressed proteins and predicted kinases in this study can be used as baits to identify candidate therapeutic drugs for COVID-19. This study can serve as a solid resource for the scientific community. The data in this manuscript will serve as a starting point for future research on COVID-19-associated encephalopathy. FUNDING: This study was supported by grants from the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, the National Natural Science Foundation of China, and the Natural Science Foundation of Beijing.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Dissecting Phenotype from Genotype with Clinical Isolates of SARS-CoV-2 First Wave Variants.

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Comparison of the pathogenesis of SARS-CoV-2 infection in K18-hACE2 mouse and Syrian golden hamster models.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extrapulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using preclinical models.

Effect of severe acute respiratory syndrome coronavirus 2 infection during pregnancy in K18-hACE2 transgenic mice.

Objective: This study aimed to examine the influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on pregnancy in cytokeratin-18 (K18)-hACE2 transgenic mice. Methods: To determine the expression of hACE2 mRNA in the female reproductive tract of K18-hACE2 mice, RT-PCR was performed using the ovary, oviduct, uterus, umbilical cord, and placenta. SARS-CoV-2 was inoculated intranasally (30 µl/mouse, 1×104 TCID50/ml) to plug-checked K18-hACE2 homozygous female mice at the pre-and post-implantation stages at 2.5 days post-coitum (dpc) and 15.5 dpc, respectively. The number of implantation sites was checked at 7.5 dpc, and the number of normally born pups was investigated at 20.5 dpc. Pregnancy outcomes, including implantation and childbirth, were confirmed by comparison with the non-infected group. Tissues of infected mice were collected at 7.5 dpc and 19.5 dpc to confirm the SARS-CoV-2 infection. The infection was identified by performing RT-PCR on the infected tissues and comparing them to the non-infected tissues. Results: hACE2 mRNA expression was confirmed in the female reproductive tract of the K18-hACE2 mice. Compared to the non-infected group, no significant difference in the number of implantation sites or normally born pups was found in the infected group. SARS-CoV-2 infection was detected in the lungs but not in the female reproductive system of infected K18-hACE2 mice. Conclusion: In K18-hACE2 mice, intranasal infection with SARS-CoV-2 did not induce implantation failure, preterm labor, or miscarriage. Although the viral infection was not detected in the uterus, placenta, or fetus, the infection of the lungs could induce problems in the reproductive system. However, lung infections were not related to pregnancy outcomes.

Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice.

Several models were developed to study the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the in vivo efficacy of vaccines and therapeutics. Since wild-type mice are naturally resistant to infection by ancestral SARS-CoV-2 strains, several transgenic mouse models expressing human angiotensin-converting enzyme 2 (hACE2) were developed. An alternative approach has been to develop mouse-adapted SARS-CoV-2 strains. Here, we compared the clinical progression, viral replication kinetics and dissemination, pulmonary tropism, and host innate immune response dynamics between the mouse-adapted MA10 strain and its parental strain (USA-WA1/2020) following intranasal inoculation of K18-hACE2 mice, a widely used model. Compared to its parental counterpart, the MA10 strain induced earlier clinical decline with significantly higher viral replication and earlier neurodissemination. Importantly, the MA10 strain also showed a wider tropism, with infection of bronchiolar epithelia. While both SARS-CoV-2 strains induced comparable pulmonary cytokine/chemokine responses, many proinflammatory and monocyte-recruitment chemokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10/CXCL10, and MCP-1/CCL2, showed an earlier peak in MA10-infected mice. Furthermore, both strains induced a similar downregulation of murine Ace2, with only a transient downregulation of Tmprss2 and no alterations in hACE2 expression. Overall, these data demonstrate that in K18-hACE2 mice, the MA10 strain has a pulmonary tropism that more closely resembles SARS-CoV-2 tropism in humans (airways and pneumocytes) than its parental strain. Its rapid replication and neurodissemination and early host pulmonary responses can have a significant impact on the clinical outcomes of infection and are, therefore, critical features to consider for study designs using these strains and mouse model. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics. In line with this, thorough evaluation of animal models and virus strains/variants are paramount for standardization and meaningful comparisons. Here, we demonstrated differences in replication dynamics between the Wuhan-like USA-WA1/2020 strain and the derivative mouse-adapted MA10 strain in K18-hACE2 mice. The MA10 strain showed accelerated viral replication and neurodissemination, differential pulmonary tropism, and earlier pulmonary innate immune responses. The observed differences allow us to better refine experimental designs when considering the use of the MA10 strain in the widely utilized K18-hACE2 murine model.

Corrigendum: Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

[This corrects the article DOI: 10.3389/fimmu.2022.1055811.].

Thermophilic Filamentous Fungus C1-Cell-Cloned SARS-CoV-2-Spike-RBD-Subunit-Vaccine Adjuvanted with Aldydrogel®85 Protects K18-hACE2 Mice against Lethal Virus Challenge.

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel®'85'-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans.

Characterization of Three Variants of SARS-CoV-2 In Vivo Shows Host-Dependent Pathogenicity in Hamsters, While Not in K18-hACE2 Mice.

Animal models are used in preclinical trials to test vaccines, antivirals, monoclonal antibodies, and immunomodulatory drug therapies against SARS-CoV-2. However, these drugs often do not produce equivalent results in human clinical trials. Here, we show how different animal models infected with some of the most clinically relevant SARS-CoV-2 variants, WA1/2020, B.1.617.2/Delta, B.1.1.529/Omicron, and BA5.2/Omicron, have independent outcomes. We show that in K18-hACE2 mice, B.1.617.2 is more pathogenic, followed by WA1, while B.1.1.529 showed an absence of clinical signs. Only B.1.1.529 was able to infect C57BL/6J mice, which lack the human ACE2 receptor. B.1.1.529-infected C57BL/6J mice had different T cell profiles compared to infected K18-hACE2 mice, while viral shedding profiles and viral titers in lungs were similar between the K18-hACE2 and the C57BL/6J mice. These data suggest B.1.1.529 virus adaptation to a new host and shows that asymptomatic carriers can accumulate and shed virus. Next, we show how B.1.617.2, WA1 and BA5.2/Omicron have similar viral replication kinetics, pathogenicity, and viral shedding profiles in hamsters, demonstrating that the increased pathogenicity of B.1.617.2 observed in mice is host-dependent. Overall, these findings suggest that small animal models are useful to parallel human clinical data, but the experimental design places an important role in interpreting the data. Importance: There is a need to investigate SARS-CoV-2 variant phenotypes in different animal models due to the lack of reproducible outcomes when translating experiments to the human population. Our findings highlight the correlation of clinically relevant SARS-CoV-2 variants in animal models with human infections. Experimental design and understanding of correct animal models are essential to interpreting data to develop antivirals, vaccines, and other therapeutic compounds against COVID-19.

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

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.

SARS-CoV-2 Omicron BA.1 Challenge after Ancestral or Delta Infection in Mice.

We assessed cross-reactivity to BA.1, BA.2, and BA.5 of neutralizing antibodies elicited by ancestral, Delta, and Omicron BA.1 SARS-CoV-2 infection in mice. Primary infection elicited homologous antibodies with poor cross-reactivity to Omicron strains. This pattern remained after BA.1 challenge, although ancestral- and Delta-infected mice were protected from BA.1 infection.

Passive immunization with equine RBD-specific Fab protects K18-hACE2-mice against Alpha or Beta variants of SARS-CoV-2.

Emergence of variants of concern (VOC) during the COVID-19 pandemic has contributed to the decreased efficacy of therapeutic monoclonal antibody treatments for severe cases of SARS-CoV-2 infection. In addition, the cost of creating these therapeutic treatments is high, making their implementation in low- to middle-income countries devastated by the pandemic very difficult. Here, we explored the use of polyclonal EpF(ab')2 antibodies generated through the immunization of horses with SARS-CoV-2 WA-1 RBD conjugated to HBsAg nanoparticles as a low-cost therapeutic treatment for severe cases of disease. We determined that the equine EpF(ab')2 bind RBD and neutralize ACE2 receptor binding by virus for all VOC strains tested except Omicron. Despite its relatively quick clearance from peripheral circulation, a 100µg dose of EpF(ab')2 was able to fully protect mice against severe disease phenotypes following intranasal SARS-CoV-2 challenge with Alpha and Beta variants. EpF(ab')2 administration increased survival while subsequently lowering disease scores and viral RNA burden in disease-relevant tissues. No significant improvement in survival outcomes or disease scores was observed in EpF(ab')2-treated mice challenged using the Delta variant at 10µg or 100µg doses. Overall, the data presented here provide a proof of concept for the use of EpF(ab')2 in the prevention of severe SARS-CoV-2 infections and underscore the need for either variant-specific treatments or variant-independent therapeutics for COVID-19.

Spike protein-independent attenuation of SARS-CoV-2 Omicron variant in laboratory mice.

Despite being more transmissible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant only causes milder diseases in laboratory animals, often accompanied by a lower viral load compared with previous variants of concern. In this study, we report the structural basis for a robust interaction between the receptor-binding domain of the Omicron spike protein and mouse ACE2. We show that pseudovirus bearing the Omicron spike protein efficiently utilizes mouse ACE2 for entry. By comparing viral load and disease severity among laboratory mice infected by a natural Omicron variant or recombinant ancestral viruses bearing either the entire Omicron spike or only the N501Y/Q493R mutations in its spike, we find that mutations outside the spike protein in the Omicron variant may be responsible for the observed lower viral load. Together, our results imply that a post-entry block to the Omicron variant exists in laboratory mice.