[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Previously cloned recombinant A116 single chain fragment variable (scFv) antibody gene has been re-engineered for enhanced reactivity to Venezuelan equine encephalitis virus (VEE) successfully. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5' region of the V(L) gene of A116, corresponding to the framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shift to a consensus framework-1 amino acid sequence. Four MA116 clones (MA116-4, MA116-14, MA116-15, and MA116-16) have been analysed in detail for their reactivity to VEE antigen, and all showed varying degrees of reactivity to VEE antigen. ScFv antibody expressed by MA116-14, MA116-15, and MA116-16 clones showed three to five-fold enhanced enzyme-linked immunosorbant assay reactivity to VEE antigen over the parental A116 clone, while scFv antibody from MA116-4 was less reactive than A116 clone. MA116-15 purified scFv protein showed comparable reactivity to the parental 1A4A-1 monoclonal antibody in recognizing VEE antigen. Sequence analysis revealed that only MA116-15 had incorporated the three intended base insertions. The varying degrees of reactivity of MA116 clones are discussed in light of their molecular changes.
[show abstract][hide abstract] ABSTRACT: Previously, the complete genome of western equine encephalitis virus had been cloned and sequenced. This paper describes mammalian expression vectors pCXH-3 and pVHX-6, in which expression of the structural genes of western equine encephalitis virus have been placed under the control of the mammalian CMV promoter. When pCXH-3 or pVHX-6 is expressed using a cell-free transcription/translation system, in vitro, authentic structural proteins of western equine encephalitis virus are produced as verified by reactivity with monoclonal antibodies developed to western equine encephalitis virus. These vectors can also be complexed with liposomes and administered to mammalian cell culture. The viral envelope proteins were functionally expressed, as determined by histochemical staining with monoclonal antibodies which recognize WEE antigens. In addition, when pCXH-3 or pVHX-6 is administered intraepidermally and intramuscularly% to mice, a protective immune response is induced. Immunized mice had a significantly reduced risk of infection, against a subsequent intranasal challenge with western equine encephalitis virus. Development of a DNA vaccine to western equine encephalitis virus is promising. In a similar manner, DNA vaccines to related alphaviruses (Venezuelan and eastern equine encephalitis viruses) could also be developed.