ABSTRACT
The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.
ABSTRACT
A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His(6)-PSP was 1.5 mg/g biomass.
Subject(s)
Protein Conformation , Serine Endopeptidases/isolation & purification , Serratia/enzymology , Substrate Specificity , Amino Acid Sequence , Base Sequence , Chromatography, Affinity , Chromatography, Ion Exchange/methods , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Molecular Weight , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serratia/genetics , Trypsin Inhibitors/pharmacologyABSTRACT
A preparative method for purification of a novel protease from the psychrotolerant Gram-negative microorganism Serratia proteamaculans (PSP) was developed using affinity chromatography on BPTI-Sepharose. It yielded electrophoretically homogeneous PSP preparation of 60 kD. The PSP properties (temperature and pH stability, high catalytic efficiency) indicate that this enzyme can be defined as a psychrophilic protease. Inhibitory analysis together with substrate specificity indicates that the studied PSP exhibits properties of serine trypsin-like and Zn-dependent protease.