Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Dependence of the Ki67 Labelling Index of Selected Canine Tumours on Patient Age, Sex and Tumour Size.

Cell proliferation is a fundamental criterion in the assessment of malignant progression of many tumours and an essential parameter in several grading schemes. However, proliferation may be dependent on patient age and other variables, as shown in normal tissues, cultured cells and human neoplasms. We thus hypothesized that age or other patient or tumour-related parameters might generally affect proliferation in canine neoplasms, which might be of value for optimizing prognostic algorithms. We performed linear regression analyses to associate age, sex and tumour size with digitally quantified immunohistochemical Ki67 labelling indices (Ki67-LIs) of 495 canine tumours, including cutaneous mast cell tumours (MCTs, n = 70), soft tissue sarcomas (n = 61), plasmacytomas (n = 86), trichoblastomas (n = 62) and perianal gland adenomas (PGAs, n = 95) as well as testicular interstitial (n = 65) and Sertoli cell tumours (n = 56). In MCTs, the Ki67-LI increased 1.13-fold per year of age (P <0.05) in bitches but not in males. Conversely, in PGAs it rose 1.10-fold per year in males (P <0.05) while it decreased 0.95-fold in bitches (P = 0.37). Only in MCTs and PGAs was the Ki67-LI associated with tumour size, albeit in oppositional directions (MCTs: 1.26-fold per cm diameter, P <0.01; PGAs: 0.76-fold, P <0.01). No correlations were found in the other tumour types. The few sex-dependent correlations with patient age and tumour size established here indicate highly tumour-type specific mechanisms, but the diagnostic consequences are uncertain.

Hamster models of COVID-19 pneumonia reviewed: How human can they be?

The dramatic global consequences of the coronavirus disease 2019 (COVID-19) pandemic soon fueled quests for a suitable model that would facilitate the development and testing of therapies and vaccines. In contrast to other rodents, hamsters are naturally susceptible to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the Syrian hamster (Mesocricetus auratus) rapidly developed into a popular model. It recapitulates many characteristic features as seen in patients with a moderate, self-limiting course of the disease such as specific patterns of respiratory tract inflammation, vascular endothelialitis, and age dependence. Among 4 other hamster species examined, the Roborovski dwarf hamster (Phodopus roborovskii) more closely mimics the disease in highly susceptible patients with frequent lethal outcome, including devastating diffuse alveolar damage and coagulopathy. Thus, different hamster species are available to mimic different courses of the wide spectrum of COVID-19 manifestations in humans. On the other hand, fewer diagnostic tools and information on immune functions and molecular pathways are available than in mice, which limits mechanistic studies and inference to humans in several aspects. Still, under pandemic conditions with high pressure on progress in both basic and clinically oriented research, the Syrian hamster has turned into the leading non-transgenic model at an unprecedented pace, currently used in innumerable studies that all aim to combat the impact of the virus with its new variants of concern. As in other models, its strength rests upon a solid understanding of its similarities to and differences from the human disease, which we review here.

Live attenuated virus vaccine protects against SARS-CoV-2 variants of concern B.1.1.7 (Alpha) and B.1.351 (Beta).

Vaccines are instrumental and indispensable in the fight against the COVID-19 pandemic. Several recent SARS-CoV-2 variants are more transmissible and evade infection- or vaccine-induced protection. We constructed live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and showed that the lead candidate, designated sCPD9, protects Syrian hamsters from a challenge with ancestral virus. Here, we assessed immunogenicity and protective efficacy of sCPD9 in the Roborovski dwarf hamster, a nontransgenic rodent species that is highly susceptible to SARS-CoV-2 and severe COVID-19­like disease. We show that a single intranasal vaccination with sCPD9 elicited strong cross-neutralizing antibody responses against four current SARS-CoV-2 variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), and B.1.617.2 (Delta). The sCPD9 vaccine offered complete protection from COVID-19­like disease caused by the ancestral SARS-CoV-2 variant B.1 and the two variants of concern B.1.1.7 and B.1.351.

Deciphering the Role of Humoral and Cellular Immune Responses in Different COVID-19 Vaccines-A Comparison of Vaccine Candidate Genes in Roborovski Dwarf Hamsters.

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

Virus-induced senescence is a driver and therapeutic target in COVID-19.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.

Development of safe and highly protective live-attenuated SARS-CoV-2 vaccine candidates by genome recoding.

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We construct a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and assess their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses do not cause clinical symptoms but are highly immunogenic and induce strong protective immunity. Attenuated viruses replicate efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters develop no signs of disease and rapidly clear challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic.

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Neutralizing Complement C5a Protects Mice with Pneumococcal Pulmonary Sepsis.

BACKGROUND: Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. METHODS: The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). RESULTS: In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. CONCLUSIONS: Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.