Natural peptidoglycan nanoparticles enable rapid antigen purification and potent delivery of plant-derived vaccines

Plant molecular farming is a promising platform for biopharmaceutical production, however, downstream processing remains a challenge due to cost and complexity. In this study, we present natural peptidoglycan nanoparticles (NPNs) derived from Gram-positive lactic acid bacteria as a novel tool for plant-based vaccine purification and delivery. Sequential treatment with trichloroacetic acid and trypsin effectively reduced NPN size, removing residual host subcellular constructs and proteins while preserving protein-binding capacity. Optimizing trimeric protein anchors and trimerization elements for plant-based expression enabled protein binding at low temperatures, minimizing proteolytic degradation. NPNs conjugated with plant-derived hemagglutinin elicited strong humoral immune responses in mice. Additionally, NPNs enhanced the retention of GFP at the injection site and supported efficient polyclonal antibody generation. These findings establish NPNs as a versatile platform for plant-based recombinant vaccine purification and delivery.