Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In the present invention, genes encoding protective antigens of unrelated bacterial, viral, parasitic, or fungal pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using only low copy expression plasmids, expression of foreign proteins is accomplished using both low copy expression plasmids in conjunction with chromosomal integrations within the same live vector. This strategy compensates for the inherent disadvantage of loss of gene dosage (versus exclusive plasmid-based expression) by integrating antigen expression cassettes into multiple chromosomal sites already inactivated in an attenuated vector.
