Development and characterization of dengue virus (DENV)-based RNA vector

Abstract

Despite the prevalent use of mRNA vaccines, from protein replacement therapy to infectious diseases and cancer, their accessibility remains limited, particularly to low- and middle-income countries. Here, the development and in vitro characterization of a DENV-based RNA vector (DV-RNA) is described, which can overcome the issue of mRNA vaccine equity. In particular, this study focused on RNA translation, immunostimulation and in-water stability, and compared their performance to that of reference RNAs. In vitro transcribed RNAs expressing nanoluciferase were transfected into cell lines using in-house cationic liposomes. Translation levels were assessed using nanoluciferase assays, while immune response were evaluated using quantitative polymerase chain reaction (qPCR). Translation screening of various DV-RNAs in HEK293T and THP-1 cells yielded one likely candidate, d1. Unexpectedly, additional elements, such as IRES or poly(A) tails, hindered DV-RNA translation and replication. Unfortunately, immunostimulation studies were inconclusive due to wide variation in data. It was observed that DV-RNA, d1, was quite stable in water when stored at 4°C, -30°C or -85°C for up to 4 weeks, but it was not comparable to other RNAs due to unreliable testing. Overall, the observed translational efficiency of these first-generation DV-RNA vectors highlight their promising potential as an RNA vector platform. While RNA immunostimulation is influenced by a complex interplay of factors that go beyond nucleoside modifications, and warrants further investigation, this study ultimately demonstrates the feasibility of DV-RNA technology and its potential to contribute to local vaccine equity.