The development of preventive or therapeutic recombinant vaccines and the generation of serodiagnostic assays for infectious diseases depend essentially on the availability of molecularly defined antigens. A major bottleneck for the identification of suitable target antigens for many pathogens is the isolation of sufficient amounts of material for subsequent genomic or proteomic screening. Applying a highly efficient expression cloning strategy to the human pathogens vaccinia virus (VV) and Chlamydia pneumoniae (CP), we demonstrate that sub-nanogram amounts of isolated nucleic acids can be utilized to determine comprehensive sets of immunodominant antigens. Remarkably, the approach not only confirmed the immunogenicity of previously reported antigens but also disclosed novel vaccine candidates conserved in orthopoxviruses, including antigenic envelope proteins and immunodominant CTL epitopes. Moreover, as illustrated for CP infection, we show that a panel of novel antigens can be readily selected from the initially discovered pool to build up pathogen-specific seroassays. The established approach is rapid, making it an attractive procedure for the comprehensive dissection of immunomes of known human pathogens and newly emerging infectious agents.