Improved uptake of plant-derived LTB-linked proteins in carp gut and induction of specific humoral immune responses upon infeed delivery.

Oral vaccination of fish is an effortless and stress free immunisation method which can be used for almost any age. However, vaccination via the mucosal route does have disadvantages. For example, the vaccine may induce tolerance and has to be protected to escape digestion. Also the vaccine should be efficiently delivered to immune-competent cells in the gut or other lymphoid organs. In addition, it should be cost effective. Here we present a novel fish vaccination model using potato tubers as vaccine production and delivery system. The model vaccines discussed here include fusion proteins consisting of a gut adhesion molecule (LTB) and a viral peptide or green fluorescent protein (GFP) expressed in potato tubers. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide or GFP functions as model vaccine antigen provoking the induction of an immune response. We demonstrate that fusion to LTB facilitates an elevated uptake of the model vaccines in carp gut mucosa. The plant-derived fusion proteins also elicit a specific systemic humoral immune response upon oral application of crude tuber material incorporated into a standard dietary feed pellet. The data presented here show the promising potentials of the plant as a production system for oral vaccines in aquaculture and feed mediated immunisation of fish.

Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein.

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.