ABSTRACT
The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.
Subject(s)
Antibodies, Neutralizing/physiology , Antibodies, Viral/physiology , Enterovirus A, Human/immunology , Enterovirus Infections/immunology , Immunoglobulin G/physiology , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/isolation & purification , Cell Line, Tumor , Enterovirus Infections/virology , Host-Pathogen Interactions , Humans , Immunoglobulin G/isolation & purification , Neutralization TestsABSTRACT
Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). EF is bacterial adenylate cyclase which, upon activation by its eukaryotic cofactor, calmodulin, causes a rapid increase in the intracellular cAMP level of host cells. EF can reduce the protective ability of host animal. In order to further research the mechanism of anthrax toxin, the expression plasmid was constructed and the structural gene for anthrax edema factor (EF) was expressed in Escherichia coli. Recombinant EF (rEF) was purified to homogeneity by a three-step procedure involving metal chelating affinity chromatography, cation-exchange chromatography and gel chromatography. From 1 liter of culture, 5mg of biologically active EF was easily purified. Rabbits were immuned with rEF, anti-EF antibodies were prepared and can neutralize rEF in cells. Tests in vitro proved rEF have good biological activity. rEF can competed the binding regions of PA with rLF in J774A.1 and CHO cells. rEF and rLF can restrain each other by competition. The successful expression of rEF has placed a solid foundation for the research on toxicity mechanism of EF, and screening for inhibitors against EF.
Subject(s)
Adenylyl Cyclases/biosynthesis , Bacillus anthracis/genetics , Adenylyl Cyclases/genetics , Adenylyl Cyclases/immunology , Adenylyl Cyclases/isolation & purification , Animals , Antigens, Bacterial , Bacillus anthracis/immunology , Bacterial Toxins , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Escherichia coli/genetics , Mice , Neutralization Tests , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purificationABSTRACT
An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Subject(s)
Anthrax Vaccines/immunology , Antigens, Bacterial/genetics , Bacterial Toxins/genetics , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/toxicity , Bacterial Toxins/chemistry , Bacterial Toxins/immunology , Bacterial Toxins/toxicity , Base Sequence , Mice , Molecular Sequence Data , Plasmids , Rabbits , Recombinant Proteins/immunology , Vaccines, Synthetic/immunologyABSTRACT
The gene encoding anthrax lehtal factor (LF) was cloned into a secretory expression plasmid and then expressed in periplasmic space of E. coli. The recombinant LF (rLF) expressed was about 4% of the total proteins in E. coli. About 3 mg electrophoresis purity rLF could be obtained after the purification of 1 liter culure using ion exchange chromatography and gel filtration. The result of sequencing assay showed that the N-terminal amino acid sequence of rLF was identical to the N-terminal sequence of natural LF. In vitro toxicity analysis also shows that rLF has an excellent biological activity. The successful expression of rLF has placed a solid foundation for the research on toxicity mechanism of LF, developing new anthrax vaccines, and screening for inhibitors against anthrax toxin.
