The potential of building multi-cytotoxic T lymphocyte (CTL) epitope antigens in combination with the nucleic acid immunization technology is explored for development of acquired immunodeficiency syndrome (AIDS) and malaria vaccines. A novel minimal vector pTH for direct gene transfer was constructed for efficient expression of vaccine antigens and used as a vehicle for human immunodeficiency virus (HIV)- and Plasmodium falciparum-derived polyepitope genes. Two murine epitopes were included into these constructs to allow for testing of vaccine immunogenicity in small animals. The results showed that a single DNA injection generated CTL responses in all 15 vaccinated mice. The elicited CTL precursor frequencies were estimated in an interferon-gamma (IFN-gamma)-based ELISPOT assay and found to be an average of 300 (range 4-1346) peptide-responding cells per 10(6) splenocytes.