Immunostimulatory nucleic acid nanoparticles establish antiviral state to inhibit viral infection and replication.

Establishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs), identified as potent innate immune activators, for their ability to induce protective antiviral states. By mimicking pathogen-associated molecular patterns, iNANPs engage intracellular pattern recognition receptors to stimulate type I and III interferon responses. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). These viruses vary in their mechanisms of innate immune activation and evasion, providing a robust system to assess iNANP activity. Our results demonstrate that iNANPs dramatically restrict viral infection via induction of a robust IFN response, establishing an antiviral state that impairs replication of all tested viruses. This study highlights the potential of iNANPs as a broad-spectrum antiviral prophylactic platform.IMPORTANCEEstablishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs) identified as potent innate immune activators for their ability to induce protective antiviral states. The highly modular design and tunable physicochemical properties of iNANPs with defined architectures and compositions can be tailored to engage specific innate immune sensors. This same modularity allows a seamless "plug-and-play" integration of diverse therapeutic nucleic acids, as well as other therapeutics and small molecules, directly into the iNANP structure. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). Our results demonstrate that iNANPs significantly restrict viral infection, highlighting their potential as a broad-spectrum antiviral prophylactic platform.