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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptorbinding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.

The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10–104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

This study examines the efficacy, bacterial load, and humoral response of extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to Francisella tularensis subsp. holarctica (strain LVS) or to the highly virulent F. tularensis subsp. tularensis (strain SchuS4). A delay in onset of both antibiotic treatments allowed the rescue of all LVS-infected animals. However, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals.

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Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium Bacillus anthracis . We previously demonstrated that disruption of htrA gene, encoding the chaperone/protease HtrA BA (High Temperature Requirement A of B. anthracis ) results in significant virulence attenuation, despite unaffected ability of Δ htrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis Δ htrA strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the htrA gene was suggested to reflect the susceptibility of Δ htrA mutated strains to stress insults encountered in the host indicating that HtrA BA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrA BA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrA BA . By inspecting the phenotype exhibited by Δ htrA strains trans -complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrA BA , as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrA BA is essential for its N-terminal autolysis and subsequent release into the extracellular milieu , while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrA BA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrA BA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrA BA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrA BA amongst bacterial serine proteases of the HtrA family.

Methyl jasmonate (MJ) acts both in vitro and in vivo against various cancer cell lines. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway results in decreased susceptibility to cytotoxic agents in many types of cancer cells. We found a strong inverse correlation between the basal level of phospho-Akt (pAkt) and the sensitivity to MJ among sarcoma cell lines. Nevertheless, levels of pAkt increased in two sarcoma cell lines, MCA-105 and SaOS-2, after MJ treatment. Treatment of both cell lines with PI3K/Akt pathway inhibitors in combination with MJ resulted in a synergistic cytotoxic effect. Moreover, cells transfected with a constitutively active Akt were less susceptible to MJ-induced cytotoxicity in comparison with cells transfected with an inactive form of Akt. Taken together, these data suggest that the increase in pAkt after treatment with MJ played a protective role. Because it has been shown that the antiapoptotic effects of Akt are dependent on glycolysis, we examined the role of glucose metabolism in activation of Akt and the subsequent resistance of the cell lines to MJ. 2-Deoxy-d-glucose, a glycolysis inhibitor, decreased the levels of pAkt and was able to attenuate the MJ-induced elevation in pAkt. Accordingly, the presence of glucose attenuated MJ-induced cytotoxicity. Moreover, treatment with 2-deoxy-d-glucose in combination with MJ resulted in a synergistic cytotoxic effect. In conclusion, the PI3K/Akt pathway plays a critical role in the resistance of MCA-105 and SaOS-2 sarcoma cell lines toward MJ-induced cytotoxicity.