Recombinant Protein Nanoparticles for Dual-Stage Inhibition of SARS-CoV‑2 Infection

Recombinant proteins exhibit significant promise as antiviral therapeutics. Here, we present a nanotherapeutic platform that, owing to its unique ability to self-assemble, can present multiple functionalities at the liquid-liquid interface. We genetically modified the sequence of an amphiphilic plant-derived protein called oleosin to induce spontaneous self-assembly into micellar nanoparticles. Using SARS-CoV-2 as a model, we demonstrate the potential of a recombinant protein nanoparticle to inhibit two different stages of viral infection. Oleosin variants were engineered to present miniproteins 1) that block the SARS-CoV-2 spike protein binding with human ACE2 receptors and 2) that inhibit virus-cell membrane fusion. Micellar nanoparticles assembled from these proteins are multivalent and bifunctional and achieved potent inhibition of the virus with an IC₅₀ of less than 8 nM, which was more than an order of magnitude lower than that from monofunctional particles. This strategy demonstrates a promising path for developing a single protein nanoparticle capable of inhibition of multiple stages of a viral infection using solely recombinant techniques.