Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines.

The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector. The selected DNA vaccine candidates were purified from E. coli DH5α and transfected into Chinese hamster ovary cells. The expression of the antigenic peptides was measured in cells along post-transfection time, through the fluorescence intensity of the reporter GFP. The lipofection efficiency of aha-pVAX-GFP was highest after 24h incubation. Formulated PLGA-chitosan nanoparticle/plasmid DNA complexes were characterized in terms of size, size distribution and zeta potential. Nanocomplexes with average diameters in the range of 150-170nm transfected in a similar fashion into CHO cells confirmed transfection efficiency comparable to that of lipofection. DNA entrapment and further DNase digestion assays demonstrated ability for pDNA protection by the nanoparticles against enzymatic digestion.

Toll-like receptors (TLRs) in aquatic animals: signaling pathways, expressions and immune responses.

The innate system's recognition of non-self and danger signals is mediated by a limited number of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are single, non-catalytic, membrane-spanning PRRs present in invertebrates and vertebrates. They act by specifically recognizing PAMPs of a variety of microbes and activate signaling cascades to induce innate immunity. A large number of TLRs have been identified in various aquatic animals of phyla Cnidaria, Annelida, Mollusca, Arthropoda, Echinodermata and Chordata. TLRs of aquatic and warm-blooded higher animals exhibit some distinctive features due to their diverse evolutionary lineages. However, majority of them share conserve signaling pathways in pathogen recognition and innate immunity. Functional analysis of novel TLRs in aquatic animals is very important in understanding the comparative immunology between warm-blooded and aquatic animals. In additions to innate immunity, recent reports have highlighted the additional roles of TLRs in adaptive immunity. Therefore, vaccines against many critical diseases of aquatic animals may be made more effective by supplementing TLR activators which will stimulate dendritic cells. This article describes updated information of TLRs in aquatic animals and their structural and functional relationship with warm-blooded animals.

Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms.

The basal position of fish in vertebrate phylogeny makes them very attractive for genomic and functional comparative immunity studies. Adaptive immunity arose early in vertebrate evolution, 450 million years ago between the divergence of cyclostomes and cartilaginous fish. The fundamental immune molecules, which include Ag-recognizing lymphocytes, immunoglobulins (Abs and Ig-family TCR), MHC products, and recombination-activating (RAG) 1 and 2 genes and the recombination mechanisms (cause of diversity in TCRs and Igs) are similar in fish and mammals. These molecules and their immune response mechanisms unravelled the primordial vertebrate immune system repertoire and adaptive radiations. Moreover, screening of animal models like zebrafish has a great importance to discover genes involved in T cell development, thymic organogenesis, and in immunity to infections. The zebrafish model may also be useful for cancer research due to its various features like rapid development, tractable genetics, ease in in vivo imaging and chemical screening.