Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.

BACKGROUND: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. RESULTS: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. CONCLUSION: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

PanG, a new ketopantoate reductase involved in pantothenate synthesis.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2.

Elevated levels of cytokines IL-1ß and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1ß released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1ß release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1ß release. After release, IL-1ß stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1ß secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1ß and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like ß-Coronaviruses.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like ß-coronavirus drugs.

Host kinase CSNK2 is a target for inhibition of pathogenic ß-coronaviruses including SARS-CoV-2.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-ß-coronavirus drugs.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6, that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

TARGETED DOWN REGULATION OF CORE MITOCHONDRIAL GENES DURING SARS-COV-2 INFECTION.

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19.

The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit.

The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.