Construction and Evaluation of Polyvalent Genetically Engineered Vaccine Candidates for VEE.
Annual rept. 30 Jun 94-29 Jun 95,
NORTH CAROLINA UNIV AT CHAPEL HILL
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Our approach to development of an improved live attenuated vaccine for Venezuelan equine encephalitis virus VEE is based on full-length cDNA clones that have been altered by site-directed mutagenesis to contain multiple attenuating mutations. Transfection of cultured cells with in vitro RNA transcripts of these clones produces attenuated progeny virus. Clones containing lethal mutations in the PE2-cleavage signal were the source for attenuated viable second-site revertants. These double mutants were characterized with respect to their physical properties, virulence phenotypes and immunogenicity to assess their utility as vaccine candidates. They were highly attenuated even when inoculated intracerebrally into CD-1 mice. Growth studies in cultured baby hamster kidney BHK cells and chicken embryo fibroblasts CEFs showed slower growth rates in CEFs than in BHK cells. Serial passage in BHK cells indicated that cell-adapted variants of these mutants could be selected. A reconstitution experiment suggested a useflil assay for the presence of virulent revertants in vaccinated animals. A single subcutaneous inoculation of an attenuated mutant appeared to completely protect the nasal epithelium from virus invasion, possibly by inducing high levels of anti-VEE mucosal IgA antibody.
- Genetic Engineering and Molecular Biology