Preexisting inter-serotype immunity drives antigenic evolution of dengue virus serotype 2

Dengue virus (DENV) infects roughly 400 million people annually, causing febrile and hemorrhagic disease. While preexisting inter-serotype immunity (PISI) provides transient protection, it may drive severe disease over time. PISI's impact on virus evolution, however, is less understood. Retrospective epidemiological analyses suggest that PISI may drive DENV evolution. Using in vitro directed evolution, we explored how DENV2 evolves in the presence of DENV3/4 convalescent serum. Two post-passaging mutations (E-I6M and E-N203D) were then studied for fitness effects in mammalian and insect hosts and immune escape. E-I6M resisted neutralization, altered fitness in mammalian cell culture models, and had no effect in Aedes albopictus mosquitoes. E-N203D showed no change in neutralization sensitivity, reduced fitness in a DENV-naïve epithelial model, and no effects in the other models. These results align with surveillance data, where E-I6M emerged and disappeared, while E-203D and E-203 N cocirculate, thus suggesting that PISI can drive DENV evolution.