Functional studies and synchrotron FTIR biochemical signatures reveal the potential of protein nanoparticles as a VHS...

As an innovative strategy towards new biomaterials for fish vaccine development, we have generated the C-terminal half of the viral haemorrhagic septicaemia virus (VHSV) G protein as nanostructured bacterial inclusion bodies (IBs). IBs offer a slow release of biologically active, native and native-like proteins from a protective scaffold based on a nontoxic amyloid network. These nanoscale materials are an attractive type of vaccine design for aquaculture, being cheap, scalable and stable in vivo without the need for encapsulation, unlike soluble proteins. The bacterial remnants carried in IBs, such as lipopolysaccharide, are safe for fish and act as immunostimulants. Here we tested VHSV-G fragment-based protein nanoparticles in a range of scenarios to ascertain cellular uptake, metabolic changes and immunogenicity. Trout (Oncorhynchus mykiss) macrophages, in the first line of defence against infections, uptake the particles, resulting in impacts on global cell biochemical signatures measured by synchrotron FTIR. These changes were similar to those observed using inactivated VHSV virus. In a trout VHSV infection model, fish immunized with the developed nanoparticles raised specific anti-VHSV IgM antibodies, detected by ELISA. Among these, neutralizing antibodies were present, shown by a viral neutralization assay in Epithelioma Papulosum Cyprini (EPC) carp cell line. Further, the anti-VHSV IgM antibody titre increased significantly in the vaccinated group post VHSV infection, compared to sham-vaccinated fish. We therefore show that viral proteins, nanostructured as IBs, can elicit specific, functional anti-viral antibodies in fish and also can mimic in vitro the metabolic signatures associated to viral stimuli. All together, these data demonstrate the potential of this strategy for vaccine development.