Oral vaccination can offer a painless and convenient method of vaccination. Furthermore, in addition to systemic immunity it has potential to stimulate mucosal immunity through antigen-processing by the gut-associated lymphoid tissues. In this study we propose the concept that pollen grains can be engineered for use as a simple modular system for oral vaccination. We demonstrate feasibility of this concept by using spores of Lycopodium clavatum (clubmoss) (LSs). We show that LSs can be chemically cleaned to remove native proteins to create intact clean hollow LS shells. Empty pollen shells were successfully filled with molecules of different sizes demonstrating their potential to be broadly applicable as a vaccination system. Using ovalbumin (OVA) as a model antigen, LSs formulated with OVA were orally fed to mice. LSs stimulated significantly higher anti-OVA serum IgG and fecal IgA antibodies compared to those induced by use of cholera toxin as a positive-control adjuvant. The antibody response was not affected by pre-neutralization of the stomach acid, and persisted for up to seven months. Confocal microscopy revealed that LSs can translocate in to mouse intestinal wall. Overall, this study lays the foundation of using LSs as a novel approach for oral vaccination.

Pollen grains and plant spores have emerged as a novel biomaterial for a broad range of applications including oral drug and vaccine delivery, catalyst support, and removal of heavy metals. However, before pollens can be used, their intrinsic biomolecules, which occupy a large part of the pollen inner cavity must be removed not only to create empty space but because they have potential to cause allergies when used in vivo. These intrinsic materials in the pollen core can be extracted through a chemical treatment to generate clean pollen shells. The commonly used method involves a series of sequential treatments with organic solvents, alkalis, and acids to remove the native pollen biomolecules. This method, though successful for treating lycopodium (Lycopodium clavatum) spores, fails for other species of pollens such as common ragweed (Ambrosia elatior) and thus prevents widespread investigation of different pollens. Herein, we report a new chemical treatment for obtaining clean pollen shells from multiple plant species. This new method involves sequential treatment with acetone, phosphoric acid, and potassium hydroxide. Scanning electron micrographs and protein quantification have shown that the new method can successfully produce clean, intact, and hollow shells from many pollen species including ragweed, sunflower, black alder, and lamb’s quarters. These results demonstrate the broad applicability of this method to clean pollens of different species, and paves the way to start investigating them for various applications.