Modern biotechnological strategies for vaccine development in aquaculture - Prospects and challenges.

Advances in genomics and the gradual reduction of cost for technologies like whole-genome sequencing have provided exciting opportunities for developing modern biotechnological-based vaccines in aquaculture. This systemic review describes the prospects and challenges of implementing these high-tech vaccines in fish species. The majority of the commercial vaccines in aquaculture utilize conventional procedures for which cost of administration, protective immunity and safety issues are the major challenges. In recent years, more efficient vaccines are being developed by adopting the advances in vaccine technology. Vaccines based on surface antigens, protein/peptide/polysaccharide subunits, recombinant DNA/mRNA/plasmids, novel antigen expression and delivery systems (bacteriophage particles, virus like particles/VLPs, recombinant yeast, mucosal vaccines), novel molecular adjuvants (IL-8, IL-12, HSPs), and encapsulation polymers and polysaccharides like chitosan nanoparticles and PLGA microcapsule were successfully developed. These biotechnology-based vaccines have proved to be very efficient in field trials, but are always in the research pipeline or as patents. Only very few of them are licensed for use, that too, in high-valued fishes like salmonids. Currently, commercial aquaculture vaccines are available for Aeromonas salmonicida, Vibrio salmonicida, Yersinia ruckeri, Vibrio anguillarum, Edwardsiella ictalurid, and for certain Betanodaviruses. Nevertheless, no registered vaccines are available for other major infectious diseases/pathogens such as viral hemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNN) and certain other betanodaviruses, channel catfish virus (CCV), gill disease bacteria, mycobacteria, flavobacterium, Edwardsiella tarda, and certain streptococci. Despite the important economic losses that the pathogens cause to aquaculture worldwide, the commercialization of vaccines remains limited due to immunological pitfalls in aquatic species, large-scale vaccination issues, unregulated use of antibiotics and chemicals, gene-based vaccine regulations and commercial viability. If attempts are to be made to develop novel delivery methods, cost-effective procedures, and relaxations in DNA vaccine regulations, biotechnology-based vaccination could circumvent the emerging disease challenges in aquaculture.

Immune responses of Cyprinus carpio induced by protein extracts of Lernaea cyprinacea Linnaeus, 1758.

Lernaea cyprinacea Linnaeus, 1758 is an ectoparasite showing widespread infections in tropical aquaculture, and the present study aimed to determine the specific immune responses against this parasite. For the experiment, whole parasite extracts were injected intraperitoneally into Cyprinus carpio Linnaeus, 1758, and samples of epidermal mucus and blood were drawn at 0, 1, 7 and 14-days post-injection (DPI). The results revealed high levels of protein, protease and lysozyme activities in the experimental fish which were injected with L. cyprinacea protein extract. In the epidermal mucus, the total protein concentration of the control fish was 460 µg/mL, and the level raised significantly to 800 µg/mL in the experimental fish. The lysozyme activity increased from 741.5 u/mL to a peak level of 1448.5 u/mL at 7DPI. The protease activity was also found elated gradually from 2.91 u/µL to 4.49 u/µL at 1 to 14 DPI. In the serum samples, the protein concentration remained steady throughout the experiment period. However, all the experimental fish displayed statistically high levels of lysozyme and protease activity, from 890 u/mL to 1220 u/mL, and 6.10 u/µL to 11.88 u/µL, respectively. In the whole blood samples, the haemoglobin content and the red blood cells (RBC) count did not show any significant change in any of the experimental groups. But, the percentage of lymphocytes showed a marginal increase from 0.47 to 0.6 in the experimental groups. Overall, the immune responses induced by L. cyprinacea protein extracts depicts a pattern of specific responses, in which the local humoral responses dominate the systemic humoral/cellular response. The results further revealed the possibility of futuristic approaches to control freshwater ectoparasites.

Transcriptomic analysis of the housefly (Musca domestica) larva using massively parallel pyrosequencing.

To explore the transcriptome of Musca domestica larvae and to identify unique sequences, we used massively parallel pyrosequencing on the Roche 454-FLX platform to generate a substantial EST dataset of this fly. As a result, we obtained a total of 249,555 ESTs with an average read length of 373 bp. These reads were assembled into 13,206 contigs and 20,556 singletons. Using BlastX searches of the Swissprot and Nr databases, we were able to identify 4,814 contigs and 8,166 singletons as unique sequences. Subsequently, the annotated sequences were subjected to GO analysis and the search results showed a majority of the query sequences were assignable to certain gene ontology terms. In addition, functional classification and pathway assignment were performed by KEGG and 2,164 unique sequences were mapped into 184 KEGG pathways in total. As the first attempt on large-scale RNA sequencing of M. domestica, this general picture of the transcriptome can establish a fundamental resource for further research on functional genomics.

Codon changed immobilization antigen (iAg), a potent DNA vaccine in fish against Cryptocaryon irritans infection.

The immobilization antigen (iAg) DNA sequence from Chiayi isolate of Cryptocaryon irritans was computationally reviewed to replace the stop codons with suitable amino acids and its GC content was intensified. The plasmid construct comprising the codon changed iAg (optiAg/optimized iAg) was successfully expressed in the bacterial strain BL21 and also in grouper fin cells (GF-1). Results of immobilization assay, ELISA and western blot of C. irritans theront and recombinant iAg by grouper antiserum against optiAg DNA indicated that the codon changed iAg retains the native conformation. The DNA vaccine construct pcDNA3.1-optiAg was encapsulated in water-oil-water triple layer emulsions measuring 19 µm diameters and was used for the immunization experiment. In trial I experiment, grouper fish were immunized twice via intramuscular injection with the pcDNA3.1-optiAg and were challenged with C. irritans at 8-day post immunization (dpi), which resulted in 46% relative percent survival (RPS). In trial II, single immunization with pcDNA3.1-optiAg boosted with recombinant iAg protein, resulted in 40% RPS. The data from this study reveal that codon change in iAg not only accomplished the expression of iAg protein in both prokaryotic and eukaryotic cell systems, but also optiAg was proved as immunogenic due to the protection it confers to the immunized fish against C. irritans infection. Hence, it is concluded that iAg can be a potent DNA vaccine in fish against infection of the ciliated protozoan, C. irritans.

A Toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection.

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interleukin-1R (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5h post-Vibrio anguillarum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum.