Abstract
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 Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) 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-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K 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-E345K 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-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.
Highlights
Dengue virus (DENV) is a vector-borne virus that is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in tropical and sub-tropical areas
We previously found that DENV-4 infectious clone viruses propagated in MRC-5 cells maintained greater genetic stability compared to viruses propagated in Vero cells, and a single E-Gly345Lys (E-E345K) mutation was detected in 50% of DENV-4 virus propagated in MRC-5 cells [9]
Results indicate that the E-E345K mutations of the DENV-4 and DENV-4D30 clones were consistent during all passages in MRC-5 cells (Fig. 1A) but were reverted to the wide type sequences in P4 Vero cells (Fig. 1B)
Summary
Dengue virus (DENV) is a vector-borne virus that is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in tropical and sub-tropical areas. Spot sequencing and TaqMan mismatch amplification mutation methods have been used to identify loss of the attenuating markers in chimeric DENV-2 PDK-53 vaccines following initial passages in Vero cells [3,7]. We previously found that DENV-4 infectious clone viruses propagated in MRC-5 (human fetal lung fibroblast) cells maintained greater genetic stability compared to viruses propagated in Vero cells, and a single E-Gly345Lys (E-E345K) mutation was detected in 50% of DENV-4 virus propagated in MRC-5 cells [9]. It was reported that three passages of the same backbone–cloned DENV-4 in fetal rhesus lung (FRhL) cells yielded a single E-Glu327Gly (E-E327G) mutation with enhanced heparin bindings, resulting in a loss of infectivity and immunogenicity in rhesus monkeys [11]. Whether the increased heparin bindings of these adaptive mutations from MRC-5, Vero and FRhL cells cause the loss of immunogenicity in rhesus monkeys is still unknown
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