Introduction
Vibriosis, a hemorrhagic septicemia caused by members of the gram-negative genus Vibrio, is one of the most prevalent diseases of cultured fish, shrimps and bivalves (1). V. anguillarum, the etiological agent of classical vibriosis in warm- and cold-water fish species, is particularly problematic, leading to high mortalities and economic losses in aquaculture (1). Although commercial vaccines have been developed against vibriosis, these are mainly injectable ones, requiring individual handling of fish, which is laborious, time-consuming and may induce stress-related mortalities (2). Present vaccine trend developments focus on alternative methods for mass delivery of antigens, including oral and immersion vaccination (2). Oral vaccine administration incorporated in feed seems to be the preferable method as it reduces fish stress and costs to the minimum and is feasible for larvae and juveniles vaccination (2). Oral vaccines are thus highly demanded by the aquaculture sector, but till today most previous attempts to obtain effective oral vaccines in fish have failed.
One possible strategy is the use of bacterial spores, extremely resistant structures with wide biotechnological applications (3), that survive passage through the gastrointestinal tract. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models (4), but, to date, such technology has not been applied against gram-negative fish bacterial diseases.
To fulfil this gap, we used B. subtilis spores as a delivery vehicle for the presentation of the OmpK immunogenic protein, an antigen shared among several Vibrio species and evaluated its efficacy in increasing survival of larvae from the model zebrafish (Danio rerio) and of juveniles from an economically important aquaculture species, the European seabass (Dicentrarchus labrax).
Materials and Methods
The genes encoding for the OmpK protein and the green fluorescence protein (GFP) were PCR amplified containing an N-terminal 6Histines-Tag and cloned into p1CSV-CotY-N and p1CSV-CotY-C plasmid vectors (5), resulting in a translational fusion to the crust protein CotY either C-or-N-terminally. Integration at B. subtilis 168 chromosome through a double-crossover recombination event, resulted in the chloramphenicol resistant strains CRS218 (CotY-H6-GFP), CRS219 (H6-GFP-CotY), CRS220 (CotY-H6-OmpK) and CRS221 (H6-OmpK-CotY). Spores of WT B. subtilis 168 and its congenic derivatives were obtained after induction of sporulation by nutrient exhaustion in DSM and purified following standard procedures. The display of the target proteins at the surface of the recombinant spores was evaluated by western blot with a commercial anti-His-Tag antibody. Strains carrying the GFP fusions were also observed under a fluorescence microscope.
The vaccination potential of OmpK-carrying spores was first evaluated in zebrafish larvae reared at 28 ºC in 6-wells plates containing egg water. At 6 dpf (days post-fertilization), larvae were treated for 2h with spores suspensions (108 CFU mL-1) of each recombinant strain carrying the OmpK-CotY fusions, or from the parental B. subtilis 168 strain. At 9 dpf larvae were challenged by immersion with 3×108 CFUs mL-1 of V. anguillarum or 1×108 CFUs mL-1 of V. parahaemolyticus. Cumulative mortalities were registered between 16-24h, and dead larvae removed and safely discarded. The experiment was independently carried out 3 times.
Next, the vaccination potential of OmpK-carrying spores was also evaluated in European seabass. Triplicate groups of 40 European seabass juveniles, were fed diets containing 1×109 spores Kg-1 feed of either CRS220 (OmpK-diet), CRS218 (GFP-diet) or from the parental B. subtilis 168 strain (CTR, control-diet). After 30 days, 10 fish from each tank of the GFP-diet and of the CTR diet were used for blood collection and subsequently used for anti-GFP antibodies detection in fish serum, using an indirect ELISA approach. Simultaneously, 60 fish from tanks of the OmpK-diet and of the CTR diet, were challenged by injection with V. anguillarum (1x106 CFU/fish), while the remaining fish from the same treatments were injected with PBS. Fish survival was followed during 7 days, with dead and moribund fish showing signs of infection daily removed and sacrificed. At the end of the challenge, the remaining fish were euthanized with an overdose of anesthetic. Survival curves were plotted using the Kaplan-Meier method and pairwise comparisons between treatments were performed with nonparametric log-rank test, at 0.05 significance level, in GraphPad Prism version 9.
Results
Both OmpK and GFP proteins were successfully displayed at the surface of the B. subtilis spore, although more efficiently in the C-terminal versions. Importantly, zebrafish survival upon challenge with V. anguillarum and V. parahaemolyticus was increased in magnitudes of 50 to 90% respectively, when previously vaccinated with OmpK-carrying spores. Further, we detected anti-GFP-antibodies in the serum of European seabass fed diets containing GFP-carrying spores and increased European seabass survival by 30% when challenged with V. anguillarum if previously fed with a diet containing OmpK-carrying spores.
Conclusions
Overall our results indicate that B. subtilis spores can be efficient antigen carriers for oral vaccine delivery in fish.
Funding: SFRH/BD/138187/2018; UIDB/04423/2020 ; UIDP/04423/2020.
References
1. Nurliyana Mohamad, Mohammad Noor Azmai Amal, Ina Salwany Md Yasin, Mohd Zamri Saad, Nurrul Shaqinah Nasruddin, Nurhidayu Al-saari, Sayaka Mino, Tomoo Sawabe. (2019). Vibriosis in cultured marine fishes: a review. Aquaculture 512.
2. Ma J, Bruce TJ, Jones EM, Cain KD. (2019). A review of fish vaccine development strategies: conventional methods and modern biotechnological approaches. Microorganisms 7(11): 569.
3.Zhang X, Al-Dossary A, Hussain M, Setlow P, Li J. (2020). Applications of Bacillus subtilis spores in biotechnology and advanced materials. Applied and Environmental Microbiology 86:e01096-20.
4. Copland Alastair, Diogo Gil R., Hart Peter, Harris Shane, Tran Andy C., Paul Mathew J., Singh Mahavir, Cutting Simon M., Reljic Rajko. (2018). Mucosal delivery of fusion proteins with Bacillus subtilis spores enhances protection against tuberculosis by Bacillus Calmette-Guérin . Frontiers in Immunology 9:346.
5. Bartels, J., Lopez Castellanos, S., Radeck, J., & Mascher, T. (2018). Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform. ACS Synth Biol, 7(2), 452-461. doi:10.1021/acssynbio.7b00285