However, the mean percentage of CD8+ T-cells in group 4 was also significantly higher than in group 1, which showed a significantly higher CD4/CD8 rate as compared to all other groups. During previous DNA vaccination studies in SPF turkeys, unformulated pcDNA1/MOMP induced significant protection against severe clinical signs and lesions, bacterial replication and excretion following an experimental Cp. psittaci infection Cyclopamine chemical structure , ,
 and . However, complete protection was never observed. One might consider whether it will ever be possible to reach complete protection, if really needed at all. Maybe the previously used DNA vaccine could already create significant economical benefits by reducing the infection pressure and bacterial spread on the farms and as such diminishing Cp. psittaci outbreaks. Nevertheless, the potency of the previously used DNA vaccine can be further improved by optimising the efficiency of plasmid transfection and ompA translation inside host cells. We therefore tried to improve the immunogenicity of the DNA vaccine by optimising the ompA sequence learn more for avian expression. Codon optimisation of ompA was performed
by Genscript corporation, increasing the codon adaptation index (CAI)  from 0.606 to 0.948. The codon-optimised ompA sequence was constructed synthetically, genetically linked to EGFP and cloned into pcDNA1, resulting in pcDNA1/MOMPopt. Subsequently, we tried to increase the transfection efficiency of the vaccine by generating pcDNA1/MOMPopt complexes using lPEI, brPEI, DOTAP/DOPE liposomes and starburst PAMAM dendrimers. SB-3CT Non-cytotoxic complexes of pcDNA1/MOMPopt with liposomes, lPEI or brPEI significantly enhanced the transfection and translation efficiency in vitro compared to pcDNA1/MOMP, while complexes generated with dendrimers gave poor transfection results. Overall, the highest transfection efficiencies were obtained when using lPEI and brPEI complexes at an N/P ratio of 8. Administration of a Cp. psittaci vaccine
to poultry should be cost effective and easy. Aerosol administration could provide a solution, as most vaccines for avian respiratory diseases (New Castle Disease, Infectious Bronchitis or Avian Pneumovirus infections) are currently administered by aerosol or spray. Additionally, it has already been demonstrated that lPEI and brPEI are suitable gene delivery systems for aerosol therapy both in vitro and in mice , , ,  and . Stability of pcDNA1/MOMPopt lPEI and brPEI polyplexes and DNA integrity during nebulisation with a Cirrus™ nebulizer (Intersurgical) was therefore assessed by measuring particle size, zeta potential and DNA concentration in addition to agarose gel electrophoresis and expression in BGM cells.