An encapsulation-free oral subunit vaccine for VHSV : Inducing robust antiviral and antibody responses

Viral haemorrhagic septicaemia (VHS) is a viral disease that causes high mortality in numerous teleost species, both farmed and wild. A commercial vaccine against the VHS virus is not yet available. Due to the difficulty and stress of early-stage injection, the oral route would undoubtedly be the optimal vaccination route in terms of animal welfare and handling costs. However, oral administration has drawbacks, primarily weak immune protection and vaccine degradation, which in most cases requires additional encapsulation. Here we present a recombinant protein subunit vaccine against VHSV that is highly stable, does not require encapsulation, and stimulates both local and systemic antiviral responses after oral intubation. Additionally, it activates the adaptive immune response, inducing the production of specific and neutralizing antibodies when administered either by intubation or incorporated into fish feed. The vaccine consists of VHSV glycoprotein G fused with an interferon-gamma (IFNγ) domain from Oncorhynchus mykiss, overexpressed in Escherichia coli and purified as a highly structured nanostructured biomaterial (nanopellets, NPs). These NPs were internalized by the rainbow trout intestinal epithelial cell line (RTgutGC) and rainbow trout head kidney macrophage (RT-HKM) cells and were fully functional triggering antiviral and inflammatory gene expression and activating the interferon signaling pathway. To evaluate whether VHSV-IFN could be absorbed in the gut and induce a local and systemic immune response, trout fry were intubated at two doses, 30 mg/kg fish and 125 mg/kg fish. Analysis of hindgut-sorted leukocytes revealed two populations-myeloid and lymphoid-the latter showing a strong antiviral response at the higher dose. Consistent with this, we observed in the head kidney (HK) of fish intubated with VHSV-IFN, a clear antiviral gene response. Interestingly, fish treated with VHSV-IFN at 125 mg/kg fish and 500 mg/kg fish administered by intubation and orally in the diet, respectively, showed an increase in specific and neutralizing antibody titres 30 days after vaccination, demonstrating its ability to induce a robust adaptive response.