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.

Introduction: Intravenous dodecafluoropentane emulsion (DDFPe) has proven ability to transport oxygen in ischemic and anemic animal models. Therapeutic effects in rabbits and pigs last at least 90 minutes, although in human studies dodecafluoropentane (DDFP) is eliminated rapidly from the blood through respiration. DDFPe neuroprotection reduces infarct volumes >80% in our rabbit embolic stroke model. Since little is known about DDFPe activity in rabbits, blood pharmacokinetics were investigated in healthy rabbits to determine dosing regimens. Hypothesis: We hypothesize that DDFP pharmacokinetics in the rabbit are similar to those in humans, with blood half-life values near 2.2±1.2 min, and clearance values near 49.6±10.8 mL/min/kg. Methods: Bilateral ear catheters, one intravenous and one intraarterial, were introduced into New Zealand White rabbits (N=8, 4.2±0.9 kg) for DDFPe administration and blood sampling, respectively. Rabbits were divided into 3 DDFPe dosage groups: 1) single bolus (0.6 mL/kg) with blood sampling for 4 hrs (N=4), 2) 15 doses (0.1 mL/kg) with sampling for 24 hrs (N=2), 3) 5 doses (0.6 mL/kg) with sampling for 7 hrs (N=2). Repeated doses of 2% w/v DDFPe were administered every 90 min. All samples were kept frozen until thawed before analysis by headspace gas chromatography-mass spectrometry. Results: An exponential regression closely approximated the blood concentration vs. time plot of single bolus sampling with an average R 2 of 0.96±0.02, thus compartmental modeling was used for pharmacokinetic analysis. The blood half-life was 1.9±0.6 min, with a clearance of 90.1±45.3 mL/min/kg. In all multi-dose rabbits, blood concentration of DDFP did not reach a steady state. The concentration vs. time data exhibited a repeated sequence of individual bolus data, with DDFP reaching undetectable levels 10 min after each dose. Conclusion: Blood elimination half-life and clearance rates of DDFPe in rabbits are comparable to humans, further indicating that the rabbit stroke model is appropriate for investigation of the neuroprotective potential of DDFPe. The time disparity between blood levels and therapeutic oxygen transport ability of DDFPe suggests multiple compartments and merits further investigation.