Safe Staphylococcal Platform for the Development of Multivalent Nanoscale Vesicles against Viral Infections

Many viruses often have closely related yet antigenically distinct serotypes. An ideal vaccine against viral infections should induce a multivalent and protective immune response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that carry different protein components, we constructed an agr locus deletion mutant of the Staphylococcus aureus strain (RN4220-Delta agr) to reduce potential toxicity. Nanoscale vesicles derived from this strain ((Delta agr)MVs) carry at least four major components that can deliver heterologous antigens. These components were each fused with a triple FLAG tag, and the tagged proteins could be incorporated into the (Delta agr)MVs. The presentation levels were (3.43 +/- 0.73)%, (5.07 +/- 0.82)%, (2.64 +/- 0.61)%, and (2.89 +/- 0.74)% of the total (MV)-M-Delta agr proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and EDIIIconB delivered by two staphylococcal components were stably embedded in the (Delta agr)MVs. Administration of such engineered (Delta agr)MVs in mice induced antibodies against all four DENV serotypes. Sera from immunized mice protected Vero cells and suckling mice from a lethal challenge of DENV-2. This study will open up new insights into the preparation of multivalent nanosized viral vaccines against viral infections.