In vitro analysis of expression vectors for DNA vaccination in horses

DNA vaccines have attracted great interest since they induce strong and lasting humoral and cellular immune response in experimental animals. Their ability to modulate the immune response and to shift it from Th2 to Th1 holds a promise for treatment of allergies and cancer. In large animals and humans DNA vaccines have, however, not lived up to this expectation. Their major drawback is low and short lived immune response. One of the reasons for this is thought to be due to limited expression of the gene product involved and few activated antigen presenting cells. It is therefore important to improve the efficasy of expression in the cells of the relevant animal. Our efforts to Th1 focus the immune response of horses by vaccinating them with vectors of pcDNA origin resulted in low immune response. The aim was to try to improve the expression from the vectors with a Kozak sequence and the cytomegalovirus (CMV) intron A. The ectopic expression of the human serum albumin gene in primary horse cells from skin, lung, duodenum and kidney was analysed. Plasmids were altered by using PCR and other conventional molecular methods, propagated in the DH5alpha strain of E. coli, harvested and purified by QIAGEN Plasmid Midi Kits. The expression of HSA was tested by transfection with lipafectamin 2000 and detection in Western blot. The vectors used were of pcDNA and pUC origin and include the CMV promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not. Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression both in COS-7 cells and primary horse cells derived from skin, lung, duodenum and kidney.