Hamster model for post-COVID-19 alveolar regeneration offers an opportunity to understand post-acute sequelae of SARS-CoV-2.

COVID-19 survivors often suffer from post-acute sequelae of SARS-CoV-2 infection (PASC). Current evidence suggests dysregulated alveolar regeneration as a possible explanation for respiratory PASC, which deserves further investigation in a suitable animal model. This study investigates morphological, phenotypical and transcriptomic features of alveolar regeneration in SARS-CoV-2 infected Syrian golden hamsters. We demonstrate that CK8+ alveolar differentiation intermediate (ADI) cells occur following SARS-CoV-2-induced diffuse alveolar damage. A subset of ADI cells shows nuclear accumulation of TP53 at 6- and 14-days post infection (dpi), indicating a prolonged arrest in the ADI state. Transcriptome data show high module scores for pathways involved in cell senescence, epithelial-mesenchymal transition, and angiogenesis in cell clusters with high ADI gene expression. Moreover, we show that multipotent CK14+ airway basal cell progenitors migrate out of terminal bronchioles, aiding alveolar regeneration. At 14 dpi, ADI cells, peribronchiolar proliferates, M2-macrophages, and sub-pleural fibrosis are observed, indicating incomplete alveolar restoration. The results demonstrate that the hamster model reliably phenocopies indicators of a dysregulated alveolar regeneration of COVID-19 patients. The results provide important information on a translational COVID-19 model, which is crucial for its application in future research addressing pathomechanisms of PASC and in testing of prophylactic and therapeutic approaches for this syndrome.

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/2 cleavage site and K986→P and V987→P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of S broadly reactive antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of hamsters.

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identified. This study investigates virus spread and associated pathology in the upper and lower respiratory tracts of Syrian golden hamsters at 4 days post intranasal SARS-CoV-2 Omicron infection, in comparison to infection with variants of concern (VOCs) Gamma and Delta as well as ancestral strain 614 G. Pathological changes in the upper and lower respiratory tract of VOC Omicron infected hamsters are milder than those caused by other investigated strains. VOC Omicron infection causes a mild rhinitis with little involvement of the olfactory epithelium and minimal lesions in the lung, with frequent sparing of the alveolar compartment. Similarly, viral antigen, RNA and infectious virus titers are lower in respiratory tissues of VOC Omicron infected hamsters. These findings demonstrate that the variant has a decreased pathogenicity for the upper and lower respiratory tract of hamsters.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters.

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.

Investigations on SARS-CoV-2 Susceptibility of Domestic and Wild Animals Using Primary Cell Culture Models Derived from the Upper and Lower Respiratory Tract.

Several animal species are susceptible to SARS-CoV-2 infection, as documented by case reports and serological and in vivo infection studies. However, the susceptibility of many animal species remains unknown. Furthermore, the expression patterns of SARS-CoV-2 entry factors, such as the receptor angiotensin-converting enzyme 2 (ACE2), as well as transmembrane protease serine subtype 2 (TMPRSS2) and cathepsin L (CTSL), cellular proteases involved in SARS-CoV-2 spike protein activation, are largely unexplored in most species. Here, we generated primary cell cultures from the respiratory tract of domestic and wildlife animals to assess their susceptibility to SARS-CoV-2 infection. Additionally, the presence of ACE2, TMPRSS2 and CTSL within respiratory tract compartments was investigated in a range of animals, some with unknown susceptibility to SARS-CoV-2. Productive viral replication was observed in the nasal mucosa explants and precision-cut lung slices from dogs and hamsters, whereas culture models from ferrets and multiple ungulate species were non-permissive to infection. Overall, whereas TMPRSS2 and CTSL were equally expressed in the respiratory tract, the expression levels of ACE2 were more variable, suggesting that a restricted availability of ACE2 may contribute to reduced susceptibility. Summarized, the experimental infection of primary respiratory tract cell cultures, as well as an analysis of entry-factor distribution, enable screening for SARS-CoV-2 animal reservoirs.

Alternatives to animal models and their application in the discovery of species susceptibility to SARS-CoV-2 and other respiratory infectious pathogens: A review.

The emergence of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inspired rapid research efforts targeting the host range, pathogenesis and transmission mechanisms, and the development of antiviral strategies. Genetically modified mice, rhesus macaques, ferrets, and Syrian golden hamsters have been frequently used in studies of pathogenesis and efficacy of antiviral compounds and vaccines. However, alternatives to in vivo experiments, such as immortalized cell lines, primary respiratory epithelial cells cultured at an air-liquid interface, stem/progenitor cell-derived organoids, or tissue explants, have also been used for isolation of SARS-CoV-2, investigation of cytopathic effects, and pathogen-host interactions. Moreover, initial proof-of-concept studies for testing therapeutic agents can be performed with these tools, showing that animal-sparing cell culture methods could significantly reduce the need for animal models in the future, following the 3R principles of replace, reduce, and refine. So far, only few studies using animal-derived primary cells or tissues have been conducted in SARS-CoV-2 research, although natural infection has been shown to occur in several animal species. Therefore, the need for in-depth investigations on possible interspecies transmission routes and differences in susceptibility to SARS-CoV-2 is urgent. This review gives an overview of studies employing alternative culture systems like primary cell cultures, tissue explants, or organoids for investigations of the pathophysiology and reverse zoonotic potential of SARS-CoV-2 in animals. In addition, future possibilities of SARS-CoV-2 research in animals, including previously neglected methods like the use of precision-cut lung slices, will be outlined.

Ferrets are valuable models for SARS-CoV-2 research.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in an ongoing pandemic with millions of deaths worldwide. Infection of humans can be asymptomatic or result in fever, fatigue, dry cough, dyspnea, and acute respiratory distress syndrome with multiorgan failure in severe cases. The pathogenesis of COVID-19 is not fully understood, and various models employing different species are currently applied. Ferrets can be infected with SARS-CoV-2 and efficiently transmit the virus to contact animals. In contrast to hamsters, ferrets usually show mild disease and viral replication restricted to the upper airways. Most reports have used the intranasal inoculation route, while the intratracheal infection model is not well characterized. Herein, we present clinical, virological, and pathological data from young ferrets intratracheally inoculated with SARS-CoV-2. Infected animals showed no significant clinical signs, and had transient infection with peak viral RNA loads at 4 days postinfection, mild to moderate rhinitis, and pulmonary endothelialitis/vasculitis. Viral antigen was exclusively found in the respiratory epithelium of the nasal cavity, indicating a particular tropism for cells in this location. Viral antigen was associated with epithelial damage and influx of inflammatory cells, including activated neutrophils releasing neutrophil extracellular traps. Scanning electron microscopy of the nasal respiratory mucosa revealed loss of cilia, shedding, and rupture of epithelial cells. The currently established ferret SARS-CoV-2 infection models are comparatively discussed with SARS-CoV-2 pathogenesis in mink, and the advantages and disadvantages of both species as research models for zoonotic betacoronaviruses are highlighted.

The Upper Respiratory Tract of Felids Is Highly Susceptible to SARS-CoV-2 Infection.

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.