Development of Plant-Based Subunit Vaccine Against Vibriosis for Use in Aquaculture

Abstract

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. Vibriosis is a deadly hemorrhagic septicemic disease that affects fish, bivalves, and crustaceans, causing considerable economic losses. There are different hurdles to controlling vibriosis such as the lack of effective vaccines, their reversion to virulent form from attenuated ones, and the development of resistance to existing antibiotics due to overuse. Secondly, for fish vaccination, methods such as injection and immersion are costly. The injection method requires expensive machinery and skilled labour while immersion vaccination requires multiple doses and a large amount of vaccine. The alternate method for fish vaccination is oral vaccination. This method is stress-free and cost-effective. The vaccine antigen expressed in plants could be used to develop a cost-effective oral vaccine as the plant-based expression system has several advantages like ease of cultivation in large areas, considerably high yield of antigens, easy storage at room temperature and low cost. A novel, secure, and stable vaccine is needed to eradicate vibriosis. In the present study, for reverse vaccinology and consequent plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen. The OmpK is conserved among many Vibrio species, thus a good candidate for broad spectrum vaccine against vibriosis. Bioinformatics tools were used to investigate the biochemical and physiochemical characteristics of the OmpK vaccine construct (OVC). OVC proved as an ideal vaccine candidate as it demonstrated immunogenic, non-allergic, and nontoxic behaviors. Other physiochemical characteristics were also satisfying and supported the OVC as a potent vaccine candidate. Molecular docking of OmpK with TLR-5 was carried out by ClusPro2. The structural analysis produced a high-quality model that interacted well with TLR-5. We stably expressed outer membrane protein K (OmpK) in Nicotiana tabacum via Agrobacterium-mediated transformation. OmpK gene was cloned using the Gateway® cloning. The Polymerase Chain Reaction (PCR) was done to confirm the proper integration of OmpK transgene in the transformed plants. The transgene copy number was calculated via qRT-PCR as two. The transgenic protein checked with Western blot showed monomeric form of OmpK. ELISA showed a maximum expression up to 0.38% of OmpK protein in the soluble fraction of total plant protein. Mice and fish were immunized with plant derived OmpK antigen, both showed significantly high Abstract Development of Plant-Based Subunit Vaccine Against Vibriosis for Use in Aquaculture xiv levels of anti-OmpK antibodies. The maximum antibody titer was recorded for subcutaneously vaccination with OmpK transgenic protein. Edible plants can be used as alternative bio-factories to produce vaccine antigens. In this study, OmpK was also transiently expressed via Agrobacterium-mediated transformation in edible plants (Spinacia oleracea L. and Lactuca sativa L.). OmpK transgene expression in the infiltrated leaves of spinach and lettuce was confirmed through PCR which showed expression of OmpK in all leaves which was diminishing with time. OmpK protein was detected through Western blotting which showed a monomeric OmpK on the 2nd to 5th day in case of lettuce. Whereas in the case of spinach, the band was detected on the 2nd to 4th day, but no OmpK band was detected on the 5th day. ELISA-based quantification of OmpK revealed maximum expression in lettuce (0.45%) and spinach (0.35%) on 3rd day post agroinfiltration. The expression of OmpK in the edible plant could help in the development of an efficient oral subunit vaccine against vibriosis. The present study is the first report of OmpK antigen expression in higher plants for the potential use as a vaccine in aquaculture against vibriosis, which could protect infection from multiple Vibrio species due to the conserved nature of OmpK antigen. Development of Plant-Based Subunit Vaccine Against Vibriosis for Use in Aquaculture INTRODUCTION