The immune responses elicited in mice, after intradermal (i.d.) immunization with plasmids encoding secreted or intracellular forms of HIV-1 nef, HIV-1 tat or C. pneumoniae omp2 proteins, respectively, were compared. To mediate secretion of these proteins the genes were fused to a heterologous signal sequence from murine heavy chain IgG. The nef- and omp2-specific antibody responses were dramatically increased when mice were inoculated with the plasmid encoding the secreted form of these proteins. In contrast, HIV-1 tat comprising an internal strong nuclear targeting sequence could not be induced to secretion and subsequently no enhanced antibody response was observed. Slight improvement of the HIV-1 nef antibody response was achieved after co-inoculation with a granulocyte-macrophage colony-stimulating factor (GM-CSF) expression vector. Further, nef-specific T-cell responses were induced after nef DNA injections, and were of Th1-like phenotype regardless of whether the nef protein was secreted or not. The system described in this study, using a plasmid vector with a strong heterologous signal sequence that mediate efficient antigen secretion in vivo, may have wide applicability for the induction of high antibody levels to normally non-secreted antigens.