Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We constructed a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome, and assessed 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 did not cause clinical symptoms, but were highly immunogenic and induced strong protective immunity. Attenuated viruses replicated efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters developed no signs of disease and rapidly cleared 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.

Prokaryotes are unicellular organisms that require sensory networks for their survival in rapidly changing habitats. In the course of evolution, transmembrane signaling systems have evolved to transmit signals from the extracellular environment across the cytoplasmic membrane into the cell. One-component signaling systems represent the oldest and simplest solution for such signal transmission, whereas two-component systems are evolutionarily younger (Ulrich et al., 2005). Although one-component systems are widely distributed among bacteria, only 3% are directly integrated into cytoplasmic membranes (Ulrich et al., 2005). A literature search revealed a non-exhaustive list of signaling molecules that includes ToxRS,

In COVID-19, the immune response largely determines disease severity and is key to therapeutic strategies. Cellular mechanisms contributing to inflammatory lung injury and tissue repair in SARS-CoV-2 infection, particularly endothelial cell involvement, remain ill-defined. We performed detailed spatiotemporal analyses of cellular and molecular processes in SARS-CoV-2 infected Syrian hamsters. Comparison of hamster single-cell sequencing and proteomics with data sets from COVID-19 patients demonstrated inter-species concordance of cellular and molecular host-pathogen interactions. In depth vascular and pulmonary compartment analyses (i) supported the hypothesis that monocyte-derived macrophages dominate inflammation, (ii) revealed endothelial inflammation status and T-cell attraction, and (iii) showed that CD4+ and CD8+ cytotoxic T-cell responses precede viral elimination. Using the Syrian hamster model of self-limited moderate COVID-19, we defined the specific roles of endothelial and epithelial cells, among other myeloid and non-myeloid lung cell subtypes, for determining the disease course.

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Bile resistance is essential for enteric pathogens, as exemplified by Vibrio cholerae, the causative agent of cholera. The outer membrane porin OmpU confers bacterial survival and colonization advantages in the presence of host‐derived antimicrobial peptides as well as bile. Expression of ompU is controlled by the virulence regulator ToxR. rpoE knockouts are accompanied by suppressor mutations causing ompU downregulation. Therefore, OmpU constitutes an intersection of the ToxR regulon and the σE‐pathway in V. cholerae. To understand the mechanism by which the sigma factor σE regulates OmpU synthesis, we performed transcription studies using ompU reporter fusions and immunoblot analysis. Our data revealed an increase in ompU promoter activity in ΔrpoE strains, as well as in a ΔompU background, indicating a negative feedback regulation circuit of ompU expression. This regulation seems necessary, since elevated lethality rates of ΔrpoE strains occur upon ompU overexpression. Manipulation of OmpU’s C‐terminal portion revealed its relevance for protein stability and potency of σE release. Furthermore, ΔrpoE strains are still capable of elevating OmpU levels under membrane stress conditions triggered by the bile salt sodium deoxycholate. This study provides new details about the impact of σE on ompU regulation, which is critical to the pathogen’s intestinal survival.

In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF 12 A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated P Y. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the P Y promoter. To better understand the role of these proteins in P Y promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJ R1 (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1P Y DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between −51 and −38 in the R1P Y promoter region. Moreover, TraJ R1 and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1P Y regions proximal to the transcription start site. Our findings were corroborated by P Y-lacZ reporter fusions with a series of site specific R1P Y promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The P Y promoter became activatable by F encoded TraJ whereas TraJ R1 lost its activation function. The R1P Y mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the −35 region is critical for P Y regulation and function.

Evolution relies on the availability of genetic diversity for fitness-based selection. However, most DNA viruses employ DNA polymerases capable of exonucleolytic proofreading to limit mutation rates during DNA replication. The relative genetic stability produced by high fidelity genome replication can make studying DNA virus adaptation and evolution an intensive endeavor, especially in slowly replicating viruses. Here, we present a proofreading impaired polymerase mutant (Y547S) of Marek’s disease virus that exhibits a hypermutator phenotype while maintaining unimpaired growth in vitro and wild-type (WT)-like pathogenicity in vivo. At the same time, mutation frequencies observed in Y547S virus populations are 2–5 fold higher compared to the parental WT virus. We find that Y547S adapts faster to growth in originally non-permissive cells, evades pressure conferred by antiviral inhibitors more efficiently, and is more easily attenuated by serial passage in cultured cells when compared to WT. Our results suggest that hypermutator viruses can serve as a tool to accelerate evolutionary processes and help identify key genetic changes required for adaptation to novel host cells and resistance to antiviral therapy. Similarly, the rapid attenuation achieved through adaption of hypermutators to growth in cell culture enables identification of genetic chances underlying attenuation and virulence, knowledge that could practically exploited, for example in the rational design of vaccines.

Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity.