Production of Tetravalent Dengue Virus Envelope Protein Domain III Based Antigens in Lettuce Chloroplasts and Immunologic Analysis for Future Oral Vaccine Development

Dengue fever is a mosquito (Aedes aegypti) -transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1-4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD-TDV), is available after many decades' efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII-1-4) in stably transformed lettuce chloroplasts. Transplastomic EDIII-1-4-expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII-1-4 antigens was demonstrated by immunoblotting, with the EDIII-1-4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII-1-4. Our in vitro gastrointestinal digestion analysis revealed that EDIII-1-4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.