Abstact

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.