SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders

Introduction The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. Objectives To assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. Methods Human primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. Results LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation. Conclusions Glycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination. Graphical abstract

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells

Background The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. Results Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. Conclusions Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

ISO 10993 Biological Evaluation of Novel Hemostatic Powder – 4SEAL®

Background:Hemostasis plays a crucial role during every surgery allowing for a bloodless operating field. Fast and effective surgery leads to a reduced risk of postoperative complications. One of the latest method for achieving homeostasis is the use of natural polysaccharide-based hemostatic powders. The study aimed to evaluate the biocompatibility according to the ISO 10993 standards of 4SEAL® Hemostatic powder.Methods:Chemical characterization (Headspace GC-MS, GC-MS, and ICP-MS), cytotoxicity, genotoxicity (MLA and AMES), endotoxin contamination, sensitization potential, intracutaneous reactivity, acute and subacute systemic toxicity with implantation, and pyrogenicity were evaluated to investigate the biocompatibility of the 4SEAL® Hemostatic powder. Studies were conducted according to ISO 10993 standards.Results:The biocompatibility requirements with accordance to ISO 10993-1 for 4SEAL® Hemostatic powder were met.Conclusions:4SEAL® Hemostatic powder is a promising new hemostatic agent with a wide range of potential applications and excellent biocompatibility.

Engineering for an HPV 9-valent Vaccine using Genomic Constitutive Over-expression and Low Lipopolysaccharide Levels in Escherichia Coli Cells

BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD­—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. ConclusionReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Endotoxin contamination of single-use sterile surgical gloves

Aim: Strict endotoxin limits are enforced for implants and catheters inserted into the body. However, no standard limit has been set for single-use sterile surgical gloves. Materials & methods: Four types of gloves sold in Japan were dipped in saline and that endotoxin levels were measured. Cytokine producing activity of gloves in blood was also measured. Results: Three of the four types of gloves showed endotoxin contamination. We also confirmed an increase in cytokine production in these gloves except one glove in which anionic surfactants was found. Conclusion: The extent to which detected endotoxins enter the body during surgery is controversial, but strict endotoxin limits need to be established.