Vaccines play a critical role in combating the COVID-19 pandemic. Future control of the pandemic requires improved vaccines with high efficacy against newly emerging SARS-CoV-2 variants and the ability to reduce virus transmission. Here we compare immune responses and preclinical efficacy of the mRNA vaccine BNT162b2, the adenovirus-vectored spike vaccine Ad2-spike and the live-attenuated virus vaccine candidate sCPD9 in Syrian hamsters, using both homogeneous and heterologous vaccination regimens. Comparative vaccine efficacy was assessed by employing readouts from virus titrations to single-cell RNA sequencing. Our results show that sCPD9 vaccination elicited the most robust immunity, including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue after challenge with heterologous SARS-CoV-2. Overall, our results demonstrate that live-attenuated vaccines offer advantages over currently available COVID-19 vaccines.

Rationale: Mechanical ventilation is life-saving but may evoke ventilator-induced lung injury. Objectives: To explore how the circadian clock modulates severity of murine ventilator-induced lung injury via the core clock component BMAL1 in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM^Bmal1-/-) or wild type control (LysM^Bmal1+/+) mice were subjected to 4 hours mechanical ventilation (34 mL/kg bodyweight) to induce lung injury. Ventilation was initiated at dawn or dusk, or in complete darkness (circadian time 0 or 12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared to dusk. Experiments at circadian time showed deterioration of pulmonary function, worsening of oxygenation and increased mortality at circadian time 0 compared to circadian time 12. Wild type neutrophils isolated at dawn showed higher activation and ROS production compared to dusk, while these day-night differences were dampened in LysM^Bmal1-/-neutrophils. In LysM^Bmal1-/-mice, circadian variations in VILI severity were dampened and VILI-induced mortality at circadian time 0 was reduced compared with LysM^Bmal1+/+mice. Conclusion: Inflammatory response and lung barrier dysfunction upon mechanical ventilation exhibit diurnal variations, regulated by the circadian clock. LysM^Bmal1-/-mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysM^Bmal1+/+mice. Our data suggest that the internal clock in myeloid cells is an important modulator of ventilator-induced lung injury.

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl ⁻ ] _i and [Ca ²⁺ ] _i ). Inhibition of the chloride ion–sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca ²⁺ ] _i , whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca ²⁺ ] _i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 ( Trpv4 ^−/− ) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae . The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.

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-3β inhibitor, CHIR99021. By RNA-seq profiling of GSK-3β knockdown organoids we identified additional candidate target genes of the inhibitor, among othersFOXM1andEGF. 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.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) bacteria are a serious global health concern due to their drug-resistance to nearly all available antibiotics, fast spread, and high mortality rate. O2afg is a major CR-Kp serotype in the sequence type 258 group (KPST258) that is weakly immunogenic in humans. Here, we describe the creation and evaluation of semisynthetic O2afg glycoconjugate vaccine leads containing one and two repeating units of the polysaccharide epitope that covers the surface of the bacteria conjugated to the carrier protein CRM₁₉₇. The semisynthetic glycoconjugate containing two repeating units induced functional IgG antibodies in rabbits with opsonophagocytic killing activity and enhanced complement activation and complement-mediated killing of CR-Kp. Passive immunization reduced the burden of acute pneumonia in mice and may represent an alternative to antimicrobial therapy. The semisynthetic glycoconjugate vaccine lead against CR-Kp expressing O2afg antigen is awaiting preclinical development.

Genomic material in wastewater provides a rich source of data for detection and surveillance of microbes. Used for decades to monitor poliovirus and other pathogens, the SARS-CoV-2 pandemic and the falling costs of high-throughput sequencing have substantially boosted the interest in and the usage of wastewater monitoring. We have longitudinally collected over 100 samples from a wastewater treatment plant in Berlin/Germany, from March 2021 to July 2022, in order to investigate three aspects. First, we conducted a full metagenomic analysis and exemplified the depth of the data by temporal tracking strains and to a certain extent also variants of human astroviruses and enteroviruses. Second, targeting respiratory pathogens, a broad enrichment panel enabled us to detect waves of RSV, influenza, or common cold coronaviruses in high agreement with clinical data. Third, by applying a profile Hidden Markov Model-based search for novel viruses, we identified more than 100 thousand novel transcript assemblies likely not belonging to known virus species, thus substantially expanding our knowledge of virus diversity. Taken together, we present a longitudinal and deep investigation of the viral genomic information in wastewater that underlines the value of sewage surveillance for both public health purposes and planetary virome research.