A Novel Efficient Piscine Oral Nano-Vaccine Delivery System: Modified Halloysite Nanotubes (HNTs) Preventing Streptococcosis Disease in Tilapia (Oreochromis sp.).

Generally, the injection method is recommended as the best efficient method for vaccine applications in fish. However, labor-intensive and difficult injection for certain fish sizes is always considered as a limitation to aquatic animals. To demonstrate the effectiveness of a novel oral delivery system for the piscine vaccine with nano-delivery made from nano clay, halloysite nanotubes (HNTs) and their modified forms were loaded with killed vaccines, and we determined the ability of the system in releasing vaccines in a mimic digestive system. The efficaciousness of the oral piscine vaccine nano-delivery system was evaluated for its level of antibody production and for the level of disease prevention in tilapia. Herein, unmodified HNTs (H) and modified HNTs [HNT-Chitosan (HC), HNT-APTES (HA) and HNT-APTES-Chitosan (HAC)] successfully harbored streptococcal bivalent vaccine with inactivated S. agalactiae, designated as HF, HAF, HCF and HACF. The releasing of the loading antigens in the mimic digestive tract demonstrated a diverse pattern of protein releasing depending on the types of HNTs. Remarkably, HCF could properly release loading antigens with relevance to the increasing pH buffer. The oral vaccines revealed the greatest elevation of specific antibodies to S. agalactiae serotype Ia in HCF orally administered fish and to some extent in serotype III. The efficacy of streptococcal disease protection was determined by continually feeding with HF-, HAF-, HCF- and HACF-coated feed pellets for 7 days in the 1st and 3rd week. HCF showed significant RPS (75.00 ± 10.83%) among the other tested groups. Interestingly, the HCF-treated group exhibited noticeable efficacy similar to the bivalent-vaccine-injected group (RPS 81.25 ± 0.00%). This novel nano-delivery system for the fish vaccine was successfully developed and exhibited appropriated immune stimulation and promised disease prevention through oral administration. This delivery system can greatly support animals' immune stimulation, which conquers the limitation in vaccine applications in aquaculture systems. Moreover, this delivery system can be applied to carrying diverse types of biologics, including DNA, RNA and subunit protein vaccines.

Novel Vaccine Development for Fish Culture Based on the Multiepitope Concept.

For the past several decades, aquaculture all around the world have been retarded by various disease outbreaks caused by many pathogens including parasites, bacteria, and viruses. Apart from being harmful to human health, the emerging diseases also dramatically affect the farm animals such as livestock and aquatic animals. To cope with this problem, one of the effective prophylactic measures is the application of vaccine. However, the traditional vaccines still have some limitations and several drawbacks; thus there is a need for the development of novel advanced vaccine such as chimeric multiepitope vaccine. Based on the current understanding of genomics and immunoproteomics together with the present bioinformatics tools, the researchers can identify the potential targeted epitopes being recognizable by the immune cells. Additionally, another critical point that should be considered for designing the chimeric multiepitope vaccine is the exposure of all those epitopes to the host organism. Thus, selecting an appropriate linker and joining each identified epitope in a suitable site can create the ideal protein structure protruding all the selected epitopes on its surface. Herein, our study would provide the fundamental platform to develop the multiepitope B-cell vaccine for the prevention and control of the aquatic animal disease starting with the epitope prediction until in vivo testing the multiepitope vaccine efficacy.

Novel Chimeric Multiepitope Vaccine for Streptococcosis Disease in Nile Tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57-76% and 17-30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.