Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays.

The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV E1 and E2. The potential of the recombinant WEEV E1 and E2 as antigens for serologic tests to detect anti-WEEV antibodies for diagnosis of WEEV infection was assessed by an enzyme-linked immunosorbent assay with anti-WEEV polyclonal antibodies obtained from the mice infected with WEEV. The anti-WEEV antibodies bound the recombinant WEEV E1 and E2 in a dose dependent manner. On the contrary, antibodies against Venezuelan equine encephalitis virus with a genetic background and a disease spectrum very similar to WEEV, did not bind to the recombinant WEEV E1 and E2. Our results suggest that the recombinant WEEV E1 and E2 possess predominant antigenicity of WEEV and have the potential to be used as antigens in immunoassays to detect anti-WEEV antibodies for serological diagnosis of WEEV infection so as to eliminate the need for preparation of cell culture-derived viral antigens, which is time-consuming, expensive, laborious, tedious, and hazardous.

Cloning and expression in E. coli of a functional Fab fragment obtained from single human lymphocyte against anthrax toxin.

Human lymphocytes derived from the blood of a donor immunized with anthrax vaccine were isolated and enriched for B-cells by Nycoprep density centrifugation. Individual anti-anthrax protective antigen (PA) B-cells were isolated by fluorescence activated cell sorting with fluorescence-labeled recombinant PA (rPA). The RNA from sorted single B-cells was extracted using plant total RNA as the carrier prior to purification by Nanoprep RNA isolation columns and then cDNA was prepared. Donor specific human Fab primer sets were developed based on rapid amplification of 5'-complementary DNA end results. Heavy chain and light chain of human Fab were amplified from the donor single B-cells by PCR. The amplified heavy and light chain were then cloned into the expression vector pASK-IBA2 and expressed in Escherichia coli (E. coli). The chains combined in vivo to form a functional Fab which was then purified as one protein. The human Fab antibodies produced by this technique were functional when tested in Western blots where the rPA was the target as well as in ELISA. This approach allowed us to obtain human Fab that retained the natural heavy and light chain pairing, which is supposed to have a high antigen-binding affinity.

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector.

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.

Efficacy of DNA vaccination against western equine encephalitis virus infection.

The efficacy of a DNA vaccine against western equine encephalitis (WEE) infection in mice was evaluated. The 26S structural region was expressed, in vitro from an internal T7 promoter using a rabbit reticulysate transcription/translation system; and from a CMV promoter after transfection into Vero cell monolayers. The proteins synthesized were reactive with anti-WEE virus (WEEV) antibodies, both in western blot analysis and histochemical staining, respectively. When the DNA vaccine plasmid, pVHX-6, was administered intraepidermally to mice, followed by challenge in a lethal mouse model, the level of protection obtained ranged from 50 to 100% amongst three strains of WEEV. Preliminary results suggest the protective immunity provided by the DNA vaccine appears to be a cell-mediated immune response, as elevated cytotoxic T lymphocyte activity was detected against the E2 protein in a T-cell proliferation assay. The efficacy results suggest a DNA vaccine may be a promising approach against WEE infection.

Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system.

A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.