Dengue Type Four Viruses with E-Glu(345)Lys Adaptive Mutation from MRC-5 Cells Induce Low Viremia but Elicit Potent Neutralizing Antibodies in Rhesus Monkeys

Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu(345)Lys (E-E(345)K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu(327)Gly (E-E(327)G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E(345)K and E-E(327)G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E(345)K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E(345)K and E(327)G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E(327)G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E(345)K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E(345)K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E(345)K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.