Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larg er animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of D NA after injection into the tibialis anterior muscle, 2) characterize cellu lar uptake and expression of DIVA in muscle and draining lymph nodes, and 3 ) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-mul dose resulted in the rapid dispersion of la beled DNA throughout the muscle. DNA was internalized within 5 min by muscl e cells near the injection site and over several hours by cells that were l ocated along muscle fibers and in the draining lymph nodes. Histochemical s taining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite s ubstantial DNA uptake by non-muscle cells. Reduction of the injection volum e to 5 mul resulted in substantially less uptake and expression of DNA by m uscle cells, and correspondingly lower immune responses against the transge ne product. However, expression and immunogenicity were restored when the 5 -mul injection was followed by electroporation in vivo, These findings indi cate that distribution and cellular uptake significantly affect the immunog enicity of DNA vaccines.