RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans and the technology, is being leveraged for rapid emergency response. In this report, we assessed immunogenicity, and, for the first time, toxicity, biodistribution and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion stabilized Spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNP). In mice, one immunization with the SAM vaccine elicited a robust Spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of Spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to Phase-1 clinical evaluation (NCT04758962).
The terminal enzyme in heme biosynthesis, ferrochelatase (E.C. 4.99.1.1), catalyzes the insertion of iron into protoporphyrin IX. Nuclear-encoded and produced in the cytoplasm, ferrochelatase is proteolytically processed upon translocation into the mitochondrion. In eukaryotes, the mature-length 42,000 Da protein is associated with the inner mitochondrial membrane, with the active site facing the mitochondrial matrix (1). The proposed catalytic mechanism (2) initially involves a metaldependent, enzyme-mediated distortion of the bound porphyrin ring allowing rapid insertion of Fe 2ϩ into the bent porphyrin (3). Distinct from the enzyme's catalytic activity, a labile 2ϩ cluster has been identified in several animal ferrochelatases including human (4), mouse (5), chicken, and frog (6). Although similar to the [2Fe-2S] 2ϩ centers found in plant ferredoxins, the ferrochelatase iron-sulfur cluster is more labile with enhanced sensitivity to degradation by nitric oxide (7). That the cluster plays no direct role part in catalysis is evidenced by the observation that bacterial, plant, and yeast ferrochelatases do not contain the metal center, and by studies showing that the redox state of the cluster is inconsequential to enzyme activity (4). However, when the cluster is disassembled, enzyme activity is lost (4, 7) and the protein readily precipitates. Hence the cluster appears to play a crucial role in maintaining protein structure in animal ferrochelatases and coupled with the sensitivity to nitric oxide, this has lead us to postulate that the cluster may serve as a regulatory NO 1 -sensor as part of an immune response (7).Site-directed mutagenesis of the five conserved cysteines closest to the COOH terminus of recombinant human ferrochelatase, identified Cys-403, Cys-406, and Cys-411 as ligands to the [2Fe-2S] cluster (8). Additionally, at this time it was proposed that certain spectroscopic anomalies and the unusual lability of the cluster may result from one noncysteinyl oxygenic ligand. The objective of the present investigation was to identify the fourth cluster ligand via additional mutagenesis experiments and by cloning, expression, and characterization of cluster-containing ferrochelatases from more distantly related organisms. We report here the first characterization of ferrochelatase from Drosophila melanogaster and, together with the new mutagenesis results with the human enzyme, these new data support the assignment of the cysteine located at position 196 in the human ferrochelatase as the fourth cluster ligand. Tracing the biological evolution of the ironsulfur cluster in ferrochelatase also provides further insight concerning the specific role of this metal center. EXPERIMENTAL PROCEDURESStrains and Cell Culture-Escherichia coli strain JM109 was used to express recombinant human ferrochelatase, mutant human ferrochelatase, and recombinant Drosophila ferrochelatase as described elsewhere (9). For the mutagenesis procedure described below, E. coli strain BMH mut-s was used to amplify the mutant plasmid. E...
Within 24 hr after oral administration of the antimalarial artesunate to rats on Day 10 or 11 postcoitum (pc), there is depletion of embryonic erythroblasts (EEbs), leading to embryo malformation and death. The proximate agent is dihydroartemisinin (DHA), the primary metabolite. We investigated the causes of EEb depletion by evaluating effects of DHA on EEbs in whole embryo culture (WEC). Rat embryos cultured starting on Day 9 pc were treated with 1 or 7 μM DHA for 24 hr starting after 19 hr of culture (∼Day 10 pc) and for 2 to 12 hr starting after 43 hr of culture (∼Day 11 pc). DHA effects indicating the depletion of EEbs were paling of the visceral yolk sac and reductions in visible blood cells, H&E-stained normal (Type II or III) EEbs, and dividing (BrdU-stained) EEbs. DHA-induced abnormal cell division was indicated by increases in symmetric and asymmetric binuclear cells. DHA-induced apoptosis was indicated by increases in TUNEL- and Caspase-3-positive cells and EEbs with fragmented nuclei. In addition, although the overall number of EEbs was decreasing, DHA caused increases in the numbers of circulating early-stage (Type I or earlier) EEbs that could not be accounted for by cell division, suggesting the release of new, less sensitive erythroblasts from the yolk sac. In summary, treatment of Day 10 or 11 pc rat embryos with DHA in WEC resulted in defective and arrested cell division in EEbs followed by apoptosis, suggesting a mechanism for their depletion after artesunate treatment in vivo.
The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.
Ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. In eukaryotes, the protein is associated with the inner surface of the inner mitochondrial membrane, and in higher animals the enzyme contains a [2Fe-2S] cluster. This cluster is highly sensitive to NO and is coordinated by four Cys residues whose spacing in the primary sequence is unique. Ferrochelatase from Drosophila melanogaster has been expressed in Escherichia coli with an amino-terminal six-histidine tag and purified to homogeneity. The protein has been crystallized with the [2Fe-2S] cluster intact. The crystals belong to space group I422, with unit-cell dimensions a = b = 158.1, c = 171.2 A and two molecules in the asymmetric unit, and diffract to 3. 0 A resolution.
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