Optimization and analysis of live attenuated DENVax-4 constructs.

摘要

Dengue virus is a flavivirus that infects millions of people every year, causing high fever and rash and resulting in death in some cases. There are four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) of dengue virus that are transmitted by the Aedes aegypti mosquito, endemic to tropical and subtropical regions of the world. Currently no vaccine for dengue fever is available. The rising number of confirmed cases and the increased habitat of Aedes aegypti increase the urgent need for a vaccine. Together with the Centers for Disease Control, Inviragen Inc. has developed a tetravalent live attenuated chimeric vaccine (DENVax) that is currently in phase II clinical trials.;DENVax is based on an attenuated DENV-2 backbone. DENV-2 strain 16681 was passaged 53 times in primary dog kidney (PDK) cells. This strain is the current DENVax-2 strain used in Inviragen's vaccine. There are nine attenuating mutations, three of which are silent. Attenuating phenotypes of DENVax-2 include temperature sensitivity, decreased plaque size, and decreased replication efficiency in mosquito cells. To generate DENVax-1, DENVax-3, and DENVax-4, prM and E genes from wild type DENV-1, DENV-3, and DENV-4 strains were cloned into the infectious cDNA clone of the attenuated DENVax-2 backbone, resulting in chimeras. Tetravalent DENVax has shown significant immunogenic responses in AG129 mice and non-human primates, and is currently in phase II clinical testing.;Data from preclinical tests showed that DENVax-4 is less immunogenic in AG129 mice and non-human primates compared to the other DENVax strains. Two projects in this thesis were completed to reengineer the current DENVax-4 strain to increase immunogenicity. The first project uses blind serial passaging of DENVax-4 first generation and reengineered DENVax-4b second generation as a method to select for strains better fit to grow in vivo. These passaged strains were tested for increased growth kinetics and immunogenicity in both AG129 mice and non-human primates. The second project uses sequencing data from the serial passaging to identify several adaptive mutations in each DENVax-4 construct. These mutations were cloned into DENVax-4 sequence to potentially optimize the strains for Vero cell growth. Three new DENVax-4 constructs were introduced, each containing a different mutation. In addition three new DENVax-4 constructs with wild type reversions of non-critical attenuating mutations were generated. Growth kinetics for all six new DENVax-4 clones were characterized, and testing was done in AG129 mice to determine neutralizing antibody titers.