Abstract
Dramatic increases in dengue (DEN) incidence and disease severity have been reported in South and South-East Asian and South American countries, in great part due to the expanded co-circulation of dengue 1-4 serotype viruses (DENV) in urban areas. There is an urgent need for a safe and effective tetravalent DEN vaccine. Several live attenuated, tetravalent DEN vaccine candidates have been generated using reverse genetic technology; these candidates provide immunity to all four DENV serotypes. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DENV-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this dissertation we first investigated two DENV-4 viruses: the infectious cDNA clone-derived DEN-4 and its derived 3’ NCR 30-nucleotide deletion mutant DEN-4Δ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared the difference between 4 and 10 passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Neurovirulence for DEN-4 and DEN-4Δ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 and DEN-4Δ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 infectious clone indicated that single point mutation of E-Q438H, E-V463L, NS2B-Q78H, and NS2B-A113T imperatively increased mouse hemorrhaging severity. Targeted mutagenesis was used to construct infectious clones, DEN-4 E-E345K and DEN-4Δ30 E-E345K, and passaged mutant viruses derived from these clones in Vero or MRC-5 cells four times. The E-E345K mutation was consistently presented in viruses recovered from MRC-5 cells, but not in viruses recovered from Vero cells. E-E345K mutant viruses showed greater attenuation and immunogenicity in mice compared to E-E327G mutant viruses. Further mutated E-E345K viruses showed increased affinities for heparan sulfate, heparin, and hyaluronic acid compared to wild type viruses, as measured by plaque reduction and heparin binding assays. Increasing electrostatic interactions between negative-charge sulfate groups in heparin or hyaluronic acid and positive-charge groups in the E protein ligands of mutant viruses may alter the virus binding to different extracellular matrix compartments, thereby attenuating DEN-4 E-E345K and DEN-4Δ30 E-E345K mutant viruses. Reduced virulence and enhanced GAG binding induced by adaptive mutation E-E345K have important implications regarding the development of live-attenuated dengue vaccines. The present study demonstrates that MRC-5 cells are suitable for the production of live-attenuated dengue vaccines and adaptive mutation E-E345K from MRC-5 cell passage is a good target for being introduced to attenuate DENV. These results provide valuable information for developing live-attenuated dengue vaccines.