Abstract
Developing adjuvant vaccines to combat rising multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections is a promising and cost-effective approach. The aim of this analysis was to construct a pDNA-CPG C274-adjuvant nano-vaccine and investigate its immunogenicity and protection in BALB/c mice. The CPG ODN C274 adjuvant was chemically synthesized and cloned into pcDNA3.1( +), and the cloning was verified using PCR and BamHI/EcoRV restriction enzyme digestion. Then, utilizing a complex coacervation approach, pDNA-CPG C274 was encapsulated by chitosan (CS) nanoparticles (NPs). TEM and DLS are used to explore the properties of the pDNA/CSNP complex. TLR-9 pathway activation was investigated in human HEK-293 and RAW 264.7 mouse cells. The vaccine's immunogenicity and immune-protective effectiveness were investigated in BALB/c mice. The pDNA-CPG C274/CSNPs were small (mean size 79.21 ± 0.23 nm), positively charged (+ 38.87 mV), and appeared to be spherical. A continuous slow release pattern was achieved. TLR-9 activation was greatest in the mouse model with CpG ODN (C274) at concentrations of 5 and 10 μg/ml with 56% and 55%, respectively (**P < 0.01). However, in HEK-293 human cells, by increasing the concentration of CpG ODN (C274) from 1 to 50 μg/ml, the activation rate of TLR-9 also increased, so that the highest activation rate (81%) was obtained at the concentration of 50 μg/ml (***P < 0.001). pDNA-CPG C274/CSNPs immunized BALB/c mice produced increased amounts of total-IgG, as well as IFN-γ and IL-1B in serum samples, compared to non-encapsulated pDNA-CPG C274. Furthermore, liver and lung injuries, as well as bacterial loads in the liver, lung, and blood, were reduced, and BALB/c mice immunized with pDNA-CPG C274/CSNPs showed potent protection (50-75%) against acute fatal Intraperitoneal A. baumannii challenge. pDNA-CPG C274/CSNPs evoked total-IgG antibodies, Th1 cellular immunity, and the TLR-9 pathway, as well as protection against an acute fatal A. baumannii challenge. Our findings suggest that this nano-vaccine is a promising approach for avoiding A. baumannii infection when used as a powerful adjuvant.