An RNA-to-RNA pipeline for rapid antiviral antibody development.

Rapid development of antibody therapeutics is often hindered by dependencies on recombinant protein production, both for antigen generation and for antibody manufacturing. To overcome these bottlenecks, we established a self-amplifying replicon RNA (repRNA) immunization and therapeutic delivery platform that enables an end-to-end RNA-to-antibody-to-RNA workflow. In this approach, alpacas are immunized with repRNA encoding virus-like particles to elicit antibody responses, peripheral blood mononuclear cells are harvested to construct phage display libraries, and broadly neutralizing heavy-chain-only antibodies (VHHs [variable heavy domain of the heavy chain]) are identified through high-throughput screening. Lead VHHs are then re-encoded into repRNA for in vivo delivery as therapeutic constructs, with engineering options for valency, potency, and serum half-life. As proof of concept, we applied this platform against enterovirus D68 (EV-D68), an emerging pathogen associated with severe respiratory disease and acute flaccid myelitis in children for which no vaccines or treatments exist. repRNA-encoded VHHs protected mice from EV-D68 challenge in both lungs and nasal cavities, and cryoelectron microscopy revealed the capsid-binding footprint and mechanism of neutralization. Together, these findings demonstrate a modular platform for rapid discovery and delivery of antiviral biologics, with EV-D68 serving as a prototype application.