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At the present time, no artificial larval diet is capable of entirely fulfilling the dietary requirements of several larval fish and crustacean species. Zooplankton live food is the basic foundation of fish larviculture, and successful rearing of fish larvae still heavily depends on an adequate supply of nutritious live food. Despite being important, the production protocols of copepods and cladocerans (Moina) are still underdeveloped in hatcheries. Rotifers and Artemia are the most commonly used live foods. However, these live foods are evidently lacking in crucial nutrient constituents. Hence, through nutrient enrichment, live food with the nutritional profile that meets the requirements of fish larvae can be produced. With the aim to maximize the effectiveness of production to optimize profitability, it is important to evaluate and improve culture techniques for the delivery of micro- and macro-nutrients as feed supplements to larvae in aquaculture systems. Bioencapsulation and enrichment are the evolving techniques in aquaculture that are commonly employed to enhance the nutritional quality of live food by integrating nutrients into them, which subsequently improves the growth, survival, and disease resistance of the consuming hosts. This review aims to highlight some of the approaches and methods used to improve the nutritional quality of live food by modifying their nutrient composition, which could have immense promise in the enhancement of aquatic animal health.

The prevalence of infectious diseases in the aquaculture industry and a limited number of safe and effective oral vaccines has imposed a challenge not only for fish immunity but also a threat to human health. The availability of fish oral vaccines has expanded recently, but little is known about how well they work and how they affect the immune system. The unsatisfactory efficacy of existing oral vaccinations is partly attributable to the antigen degradation in the adverse gastrointestinal environment of fishes, the highly tolerogenic gut environment, and inferior vaccine formulation. To overcome such challenges in designing: an easier, cost-efficient, and effective vaccination method, several encapsulation methods are being adopted to safeguard antigens from the intestinal atmosphere for their immunogenic functions. Oral vaccination is easily degraded by gastric acids and enzymes before reaching the immunological site; however, this issue can be solved by encapsulating antigens in poly-biodegradable nanoparticles, transgenic designed bacteria, plant systems, and live feeds. To enhance the immunological impact, each antigen delivery method operates at a different level. Utilizing nanotechnology, it has been possible to regulate vaccination parameters, target particular cells, and lower the antigen dosage with potent nanomaterials such as chitosan, poly D,L-lactic-co-glycolic acid (PLGA) as vaccine carriers. Live feeds such as Artemia salina can be utilized as bio-carrier, owing to their appropriate size and non-filter feed system, through a process called bio-encapsulation. It ensures the protection of antigens over the fish intestine and ensures complete uptake by immune cells in the hindgut for increased immune response. This review comprises recent advances in oral vaccination in aquaculture in terms of an encapsulation approach that can aid in future research.

Electrical neuron stimulation holds promise for treating chronic neurological disorders, including spinal cord injury, epilepsy, and Parkinson’s disease. The implementation of ultrathin, flexible electrodes that can offer noninvasive attachment to soft neural tissues is a breakthrough for timely, continuous, programable, and spatial stimulations. With strict flexibility requirements in neural implanted stimulations, the use of conventional thick and bulky packages is no longer applicable, posing major technical issues such as short device lifetime and long-term stability. We introduce herein a concept of longlived flexible neural electrodes using silicon carbide (SiC) nanomembranes as a faradic interface and thermal oxide thin films as an electrical barrier layer. The SiC nanomembranes were developed using a chemical vapor deposition (CVD) process at the wafer level, and thermal oxide was grown using a high-quality wet oxidation technique. The proposed material developments are highly scalable and compatible with MEMS technologies, facilitating the mass production of long-lived implanted bioelectrodes. Our experimental results showed excellent stability of the SiC/silicon dioxide (SiO₂) bioelectronic system that can potentially last for several decades with well-maintained electronic properties in biofluid environments. We demonstrated the capability of the proposed material system for peripheral nerve stimulation in an animal model, showing muscle contraction responses comparable to those of a standard non-implanted nerve stimulation device. The design concept, scalable fabrication approach, and multimodal functionalities of SiC/SiO₂ flexible electronics offer an exciting possibility for fundamental neuroscience studies, as well as for neural stimulation–based therapies.

Allergen-specific oral immunotherapy is a disease-modifying treatment already established for respiratory allergies and tested for the treatment of several food allergies, with promising clinical and immunological outcomes. However, orally administered allergens must pass through the gastrointestinal tract, where they are exposed to proteolytic digestion. This study describes the design of multi-layered protein bodies (PBs) in Nicotiana benthamiana as a platform for allergen encapsulation, offering potential advantages for oral immunotherapy. By co-expression of three zein variants we generated multi-layered PBs with distinct core and shell structures containing derivatives of the major fish allergen parvalbumin. The specific layering and structural integrity of the PBs were confirmed by confocal microscopy. Correlative light and electron microscopy (CLEM), combined with immunolabelling, was then used to verify the exact position of the allergens in the different layers of the PBs. In vitro experiments simulating the gastrointestinal digestion process revealed a significantly increased, layer-specific resistance of PB-encapsulated allergens compared to soluble allergens. Additionally, the uptake of PBs by human intestinal epithelial cells was simulated using Caco-2 cells. Our work provides further insight into protein storage organelle formation and novel bioencapsulation strategies to produce customized delivery vehicles, whose compartments may offer increased protection against enzymatic degradation and support prolonged persistence upon oral administration.

Summary To prevent vaccine‐associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype‐2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus‐free and cold chain‐free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low‐cost, cold chain/poliovirus‐free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non‐toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site‐specific integration of CTB‐VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9–15‐fold in chloroplasts. GM1‐ganglioside receptor‐binding ELISA confirmed pentamer assembly of CTB‐VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1‐VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB‐VP1co, formulated with plant‐derived oral adjuvants, enhanced VP1‐specific IgG1, VP1‐IgA titres and neutralization (80%–100% seropositivity of Sabin‐1, 2, 3). In contrast, IPV single dose resulted in <50% VP1‐IgG1 and negligible VP1‐IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB‐VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low‐cost solution to eradicate polio.

Summary Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

Summary The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high‐cost, limited supply of IPV, persistent circulating vaccine‐derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low‐cost cold chain‐free plant‐made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50‐fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant‐derived adjuvants after single priming with IPV significantly increased VP1‐IgG1 and VP1‐IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17–10.17 log2 titre) and seropositivity (70–90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant‐cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA‐approved antigens and adjuvants are discussed.

The ability of plants to assemble particulate structures such as virus-like particles and protein storage organelles allows the direct bioencapsulation of recombinant proteins during the manufacturing process, which holds promise for the development of new drug delivery vehicles. Storage organelles found in plants such as protein bodies (PBs) have been successfully used as tools for accumulation and encapsulation of recombinant proteins. The fusion of sequences derived from 27-kDa-γ-zein, a major storage protein of maize, with a protein of interest leads to the incorporation of the chimeric protein into the stable and protected environment inside newly induced PBs. While this procedure has proven successful for several, but not all recombinant proteins, the aim of this study was to refine the technology by using a combination of PB-forming proteins, thereby generating multi-layered protein assemblies in N. benthamiana . We used fluorescent proteins to demonstrate that up to three proteinaceous components can be incorporated into different layers. In addition to 27-kDa-γ-zein, which is essential for PB initiation, 16-kDa-γ-zein was identified as a key element to promote the incorporation of a third zein-component into the core of the PBs. We show that a vaccine antigen could be incorporated into the matrix of multi-layered PBs, and the protein microparticles were characterized by confocal and electron microscopy as well as flow cytometry. In future, this approach will enable the generation of designer PBs that serve as drug carriers and integrate multiple components that can be functionalized in different ways.

This study aimed to evaluate the immune response and the disease resistance of Pacific white shrimp (Litopenaeus vannamei) larvae administered probiotic Pseudoalteromonas piscicida 1Ub 106 CFU mL−1, prebiotic mannan-oligosaccharide (MOS) 12 mg L−1, and synbiotic (probiotic P. piscicida 1Ub 106 CFU mL−1 and 12 mg L−1 prebiotic MOS) through the bio-encapsulation of Artemia sp. The bio-encapsulation was done in the rearing medium of Artemia sp. for 4 h. The bio-encapsulated Artemia sp. was administered from mysis three to post-larvae (PL) 12. After 13 days of the feeding trial, PL13 were challenged with Vibrio harveyi MR5339 RfR (3 × 107 CFU mL−1) through immersion. The challenge test was performed in the rearing medium (30 individuals L−1) for 5 days. Total hemocyte count (THC), phenoloxidase activity (PO), and respiratory burst activity (RB) were measured before and 3 days after the challenge test, while the shrimp survival was observed until 5 days to evaluate the immune response. The expression of immune-related genes of the shrimp larvae including serine protein, peroxinectin, lipopolysaccharide, and β-1,3-glucan-binding protein was analyzed 24 h after the challenge test. The results demonstrated that THC, PO, RB, the expression of immune-related genes and the survivals of the shrimp larvae administered probiotic, prebiotic, and synbiotic were higher (p < 0.05) than that of control. The administration of probiotic P. piscisida 1Ub, prebiotic MOS, and synbiotic through the bio-encapsulation of Artemia sp. could effectively improve the immune response and the disease resistance of Pacific white shrimp larvae with the best result demonstrated by the synbiotic treatment.

There is an urgent requirement for the improvement of the white leg shrimp, Litopenaeus vannamei ; health-related indices; and immunity due to emerging diseases. Recently, probiotics have been playing an important role in L. vannamei health management. Therefore, the current pond trial was to evaluate the probiotic proficiency of commercial probiotic products of THIONIL (THIO) on the enhancement of the water, soil, growth, digestibility, survival, immune-related indices, and susceptibility of L. vannamei to infection. The study was carried out in the major shrimp culturing regions of Kavali, Nellore (Andhra Pradesh), and Ponneri (Tamil Nadu), India. Six groups (lacks/ha) of the experimental L. vannamei were allocated, including a control group (THIO 0%—untreated) and groups containing 2%, 4%, 6%, 8%, and 10% of THIO that were encapsulated with commercial feed (CP Aqua). Bioassays were performed on PLs/ shrimp at various days interval of 0, 5, 25, 50, 100, and 123th to assess productivity, anti- vibrio activity, and digestive enzyme for digestibility, histological and immunological indices, and cytotoxicity in Artemia nauplii. Significant differences were observed in the increased growth (35.71 ± 3.24 g/shrimp) and digestive parameters in 10% THIO-fed shrimp. Although in contrast to the control group, the other THIO-fed prawn groups also displayed appreciable development. The findings showed that, in comparison to the control, the gill, hepatopancreas, and stomach had reduced tissue damage with 10% THIO. Furthermore, Vibrio parahaemolyticus (0.008 × 10 4  cfu/g) and Vibrio harveyi (0.051 × 10 5  cfu/g) (vibriosis) were potentially resistant to the 10% THIO-fed group. In addition, THIO-fed prawns (10%) showed significant improvements in immune-related expresses (proPO, SOD, and SOA) in comparison to the control. In conclusion, the findings showed that the THIO treatment prawns significantly improved the quality of their water (pH, ammonia, nitrogen dioxide, hydrogen sulfide, and DO) and soil (Pb, Cr, Hg, Mg, Cu, Fe, and Ni), increased and demonstrated protection against vibrio infections. Graphical Abstract