Rapid mass production of human influenza vaccine in insects using genome editing

ABSTRACT As the recent COVID-19 pandemic has made clear, the rapid mass production of vaccines for emerging infectious diseases is of paramount importance. The use of the baculovirus/insect cell expression system for in vitro protein expression has been a game changer for vaccine production, but it also introduces challenges: transfection can be difficult, needs to be repeated with every batch, and the final product requires multiple purification steps to remove the residual baculovirus. We propose an alternative approach that could side-step the drawbacks associated with baculovirus/insect cell expression system: in vivo vaccine production in a scalable insect non-mammalian genetic model system. We aim to achieve this by using the CRISPR-Cas9 system to insert viral antigen genes for human and avian influenza into insects that are already mass produced for feed and/or waste management. Both the yellow mealworm (Tenebrio molitor) and the blow fly (Phormia regina) are promising bioreactors for manipulating protein expression in vivo due to their high metabolism and fecundity, ease of rearing, high resilience, and versatility in mass production systems (large-scale production facilities already exist for both insects). Unlike existing transgenic insect hosts such as cabbage looper larvae, both mealworms and blowflies are gregarious and can be cost effectively mass-reared at a scale of several tons per day. By using the CRISPR-Cas9 technique for recombinant protein expression to express influenza antigens in these insects, we will engineer life-stage dependent activation of the antigen production. Using existing technologies to facilitate rapid antigen protein purification, we will confirm the antigen production and characterize it. Finally, we will quantify what impact (if any) the modified genome has on phenotypes relevant to mass production and validate cost considerations for production. This work will set the stage for a steady supply of low-cost and customizable antigens, using insect biomass as an affordable and scalable bioreactor.