Date of Graduation

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Hargis, Billy M.

Committee Member

Kwon, Young Min

Second Committee Member

Thallapuranam, Suresh

Third Committee Member

Berghman, Luc R.

Fourth Committee Member

Rosenkrans, Charles F. Jr.

Keywords

Chicken; DNA aptamer; Immunological adjuvant; Vaccine

Abstract

Mineral oils and metal salts are commonly used as adjuvants to enhance acquired immunity. Recently, monoclonal antibodies (MAbs) and recombinant peptides agonist CD40 receptor have shown remarkable promise for induction of rapid and robust immune responses. Limitations of this approach MAb production costs and multiple administrations due to anti-MAb immune responses. Here we demonstrate the development of a unique and sophisticated DNA aptamer-based alternative for CD40-directed delivery of universal antigens as an alternative in chickens, and potentially other vertebrate species. This receptor, expressed by antigen-presenting cells, acts as a costimulatory molecule for activated T helper lymphocytes. After initially selecting for high affinity aptamers of independent sequence, we utilized a polymerization process where multiple aptamers were simultaneously and sequentially arranged through rolling circle amplification products (RCA-p), potentially capable of binding multiple recognition sites and causing receptor clustering. Selected sequences were demonstrated to effectively activate chicken macrophage HD11 cell line through CD40 receptors, demonstrating proof of concept in vitro. Additionally, using limited proteolysis liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), we deduced the actual amino acid sequences targeted by these aptamer products, which are essential for activation. In chapter III, we produced a biotinylated version of an effective CD40-activating RCA-p, and streptavidin-conjugated this RCA-p to a biotinylated peptide of the highly conserved type-A influenza peptide M2e. This conjugate was administered subcutaneously at either (25μg/bird) or (50μg/bird) at age 7 and 21 days. Anti-M2e IgG responses were determined at 14, 21, 28 and 35 days of age by ELISA. The RCA-p-M2e complex, at 50μg/bird, was able to induce robust seroconversion as early as 7 days post-immunization, with high antibody titer consistent through the end of the experiment. The lower dose, however, showed delayed responses and required the second administration. Taken together, these results constitute proof of concept for RCA-p-directed antigen delivery for peptide antigens and a potential tool for rapid generation of new vaccines against animal diseases.

In chapter IV we discuss PCR artifacts that develop during traditional DNA aptamers selection and proposed a repetitive thermal correction cycles [(65°C/10min, 20°C/1min)x6] of the PCR. This may improve future selection of aptamers for similar purposes.

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