Production and characterization of nervous necrosis virus vaccine antigens in wild type and CRISPR/Cas9 genome edited Nicotiana benthamiana and edible crop lettuce Lactuca sativa

Abstract

Aquaculture constitutes an important source of protein, essential omega-3 fatty acids and bioavailable micronutrients for humans. The increasing demand for aquatic food products has resulted in more intensive farming practices leading to negative impacts on aquaculture organisms and marine ecosystems. Disease outbreaks cause more than 6 billion USD loss worldwide annually and possess high risks of spreading to the wild fauna especially by viral infections. Vaccination has been proved to be effective to mitigate these problems and widely used as prophylaxis in aquaculture, but available vaccines against viral nervous necrosis (VNN) are limited currently. Plant platforms have several advantages and have been proposed as an alternative biomanufacturing method for vaccine antigens. In the present study, we report: (1) selection and design of recombinant plasmids encoding the capsid proteins (CPs) of two genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV); (2) design and optimization of plasmids for transient expression of NNV vaccine antigens in wild type Nicotiana benthamiana, CRISPR/Cas9 genome edited Nicotiana benthamiana and Lactuca sativa; (3) test of different Agrobacterium strains (LBA4404 and AGL1) for effective production of NNVCPs; (4) the expression patterns of NNVCPs over time post infiltration for different plants and cultivars; (5) successful production of NNV antigens in N. benthamiana lines and lettuce cultivars, indicating the potential of the plants as antigen producers in the development of a plant-based vaccine against VNN.