Search

We employed a directed molecular evolution approach consisting of in vitro multigene DNA recombination and screening technologies in an attempt to improve the cross-reactivity and immunogenicity of Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins E1 and E2. DNA encoding E1 and E2 from three VEEV subtypes (IA/B, IIIA, IE) and one strain each of eastern (PE-6) and western (CBA87) equine encephalitis viruses (EEEV and WEEV) were used to create libraries of recombined variants. Five different libraries were generated by recombining one or both parent glycoprotein genes. Several hundred of the recombined variants were used as naked plasmid DNA to vaccinate mice, and their serum samples were screened for the presence of antibodies in a virus-specific ELISA. Selected sera were further assayed for the presence of neutralizing antibodies by plaque reduction neutralization tests (PRNT). We found that approximately 10% of the recombined variants elicited greater levels of cross-reactive antibodies against all five viral antigens as compared to DNA vaccines expressing the parental genes. In addition, representative variants from a library in which only E2 genes were recombined elicited significantly increased neutralizing VEEV antibody titers as compared to the parent DNA vaccine and provided improved protection against aerosol VEEV challenge. These data demonstrate that in vitro, multigene DNA recombination and associated screening can lead to the selection of antigens that exhibit improved cross-reactivity, immunogenicity, and protective efficacy as compared to the wild-type proteins and suggest a broad utility for these technologies in optimizing vaccine antigens., Pub. in Vaccine, v27, p4152-4160, 2009. Sponsored in part by Defense Advanced Research Project Agency.

In an attempt to improve upon the current live, attenuated vaccine (TC-83) for Venezuelan equine encephalitis virus (VEEV), the V3526 vaccine candidate strain of VEEV was prepared by site-directed mutagenesis. Because studies indicate that virus introduced by mosquito bite may be more pathogenic than the same virus introduced by needle inoculation, there were concerns that the presence of mosquito saliva, or changes in the virus due to replication in a mosquito, might allow the virus to overcome the protective effects of prior vaccination with V3526. Therefore, we determined if hamsters vaccinated with V3526 were protected from challenge with virulent Trinidad donkey strain of VEEV. All non-vaccinated hamsters succumbed after intraperitoneal challenge or after being fed on by VEEV-inoculated Ochlerotatus taeniorhynchus. In contrast, hamsters vaccinated with V3526 were resistant to intraperitoneal challenge and infection by VEEV-infected Oc. taeniorhynchus. Therefore, the V3526 candidate vaccine elicits protection against VEEV infection by mosquito bite. In efforts to develop an improved live-attenuated vaccine for Venezuelan equine encephalitis virus (VEEV), specific mutations associated with attenuation of VEEV in rodent models were identified and inserted into a full-length cDNA clone of VEEV to produce selected isogenic strains containing one or more attenuating mutations. These were evaluated for their potential as a live, attenuated VEEV vaccine, and the V3526 strain, containing a deletion of the furin cleavage site in PE2 as well as a suppressor mutation in E1, was shown to protect mice, hamsters, and nonhuman primates challenged either by intraperitoneal (I.P.) inoculation or by aerosol. In addition, this strain replicates less efficiently in potential mosquito vectors and does not revert to virulence after multiple passages in mosquitoes. In nature, most infections are caused by the bite of an infective mosquito., Published in the American Jnl. of Tropical Medical Hygeine v78 n2 p328-332 2008.

Two live, attenuated strains of Venezuelan equine encephalitis virus (VEE), IE1150K and V3526, were administered to macaques to determine if they could elicit protection against an aerosol challenge with virulent VEE virus of the IE variety (VEEV-IE). These viruses were rescued from full-length cDNA clones of 68U201 (VEEV-IE variety) and Trinidad donkey (VEEV-IA/B variety), respectively, and both have a furin cleavage site deletion mutation and a second-site resuscitating mutation. Both vaccines elicited neutralizing antibodies to viruses of the homologous variety but not to viruses of the heterologous variety. Eight weeks after vaccination, the macaques were challenged by aerosol exposure to virulent 68U201. Macaques vaccinated with V3526 were protected as well as macaques inoculated with IE1009, the wild-type infectious clone of 68U201. However, IE1150K failed to significantly protect macaques relative to controls. V3526 has now been shown to protect macaques against both IA/B Pratt WD, Davis NL, Johnston RE, Smith JF. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 2003;21(25-26):3854-62 and IE strains of VEE viruses., Pub. in Vaccine v23 p3139-3147, 2005.