In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture

Salmonella is a major foodborne pathogen that colonizes the gastrointestinal tract of poultry, compromising bird health and productivity while posing significant public health threats. Consumption of contaminated poultry products is the leading cause of Salmonella -related illnesses in humans. Although numerous vaccination strategies exist, weak immune responses often limit the efficacy. One promising approach to address this limitation is targeting the cluster of Differentiation 40 (CD40), a costimulatory receptor expressed on antigen-presenting cells that plays a crucial role in initiating and regulating immune response. In this study, we developed two rolling circle amplification (RCA) vaccine complexes (RCA_v3 and RCA_v5) against Salmonella , each comprising a CD40-targeting component to enhance immune activation (Aptamer RCA II) and a Salmonella -binding component for antigen delivery (Anti- Salmonella RCA). We evaluated the two RCA vaccine complex designs for their ability to activate macrophages in the HD11 chicken cell line by quantifying nitric oxide production via the Griess assay. The results showed that the two RCA vaccine designs (RCA_v3 and RCA_v5) combined with formalin-killed Salmonella Enteritidis (SE) significantly (58.00 μM and 52.08 μM, respectively) enhanced macrophage activation ( p < 0.05) compared to SE alone (33.37 μM). Also, the activation levels of RCA_v3 vaccine complex were significantly higher with SE as compared with the respective counterparts without SE, RCA_v3 alone, and Aptamer RCA II (35.13 μM and 39.02 μM, respectively) ( p < 0.05). These findings offer valuable insights into developing effective vaccination strategies to control Salmonella colonization in poultry, presenting a promising approach to improving food safety and reducing public health risks associated with Salmonella infections in chickens.