Immune responses elicited by plasmid DNA vaccination can be enhanced and modulated by codelivery of cytokine-encoding plasmids. We studied whether priming of cytotoxic T lymphocyte (CTL) responses against hepatitis B surface antigen (HBsAg) by DNA vaccines injected either intramusculary or intradermally with the gene gun is enhanced by codelivery of cytokine-encoding plasmids. From a panel of tested cytokine plasmids only mouse IFNbeta, IL-15, and GM-CSF encoding plasmids showed an effect. Intradermal gene gun vaccination with 1 micro g plasmid DNA encoding intracellular HBsAg (large LS) showed enhanced CTL priming when IFNbeta, IL15, or GM-CSF encoding plasmids were codelivered; this was not observed when a DNA vaccine encoding secreted HBsAg (small S) was injected. Intramuscular injection of low (5 micro g) doses of a DNA vaccine encoding large HBsAg did not prime CTL when delivered without cytokines, with IFNbeta or IL15-encoding plasmids. However, codelivery with GM-CSF encoding plasmid DNA primed potent, specific CTL immunity detected either in a cytotoxic assay or by determining the frequency of L(d)-restricted CD8(+) T cells specifically inducible to IFNgamma production. The codelivery of GM-CSF encoding plasmids with the DNA vaccine furthermore enhanced CTL priming to a subdominant, D(d)-restricted epitope of HBsAg. The adjuvant effect of cytokine-encoding plasmids on CTL priming by DNA vaccines is thus complex and depends on: (a) the type of cytokine (or combination of cytokines) codelivered, (b) the type (intracellular vs. secreted) and dose (1-50 micro g) of the DNA vaccine, (c) the method of DNA vaccine delivery ("naked" vs. particle-coated DNA), and (d) the (intramuscular vs. intradermal) route of delivery of the DNA vaccine.