DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant.

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Chicken Egg Shell Membrane Associated Proteins and Peptides.

Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance.