Several reports have indicated that prime-boost strategies of vaccination can enhance the level of specific immunity induced by nucleic acid vaccines. The present report describes such a strategy with herpes simplex virus (HSV)-2 glycoprotein D (gD), using combinations of plasmid vector that expresses gD (pgD2) and a recombinant modified vaccinia virus Ankara vector that expresses gD (MVA-gD2). The IgG antibody response to gD and the HSV-2 neutralizing antibody response were greatest when the MVA-gD2 vector was used as the priming immunization and then was boosted with either pgD2 or MVA-gD2. Determination of the isotype profile of MVA-gD2-primed mice revealed a much broader distribution of isotypes than that seen after DNA vaccination. In addition, antigen-stimulated spleen cells from mice primed with MVA-gD2 and boosted with either MVA-gD2 or pgD2 produced higher levels of interleukin-2 and interferon-gamma than did those from pgD2-primed mice, indicating that a prime-boost immunization strategy that uses the MVA and plasmid DNA vector dramatically enhances and diversifies the humoral and cellular immune response to HSV-2 gD.