Fish immunization by duckweed biomass accumulating recombinant cyprinid herpesvirus 3 antigens induces specific immune response

Cyprinid herpesvirus-3 (CyHV-3) is highly contagious and lethal to cyprinid fish, necessitating development of sustainable vaccination strategies. Our study explores duckweed as platform for vaccine production and as vehicle for oral administration which is considered most suitable for mass vaccination of fish. Using a deconstructed potato virus X-based transient expression system, serologically recognizable recombinant CyHV-3 antigens (ORF25, ORF81, ORF136, ORF72) and fusion proteins (ORF136::T2A::ORF72, ORF25::T2A::ORF81) were accumulated in the duckweed, Landoltia punctata. The specific immune response of fish on immunization by the freeze-dried antigen-expressing duckweed biomass was proved by the serum neutralization test. Uptake of green fluorescent protein fused to the adjuvant cholera non-toxic subunit B (CTB::GFP) from duckweed biomass by fish intestinal cells was demonstrated after intubation of fish with CTB::GFP-expressing biomass. Therefore, transient expression of recombinant proteins in duckweed is a promising tool for antigen production and can facilitate the development of oral vaccines for veterinary application.