Human cytomegalovirus (CMV) is the major infectious cause of birth defects in the United States. Recent demonstration that the glycoprotein B (gB)/MF59 vaccine has 50% efficacy in protecting women against primary CMV infection is a landmark in CMV vaccine research. However, 50% efficacy would be insufficient for vaccine licensure. Thus, the challenge now is to determine what can be added to a gB-based vaccine to increase efficacy to an acceptable level. Recent work from our laboratory indicates that an important component of humoral immunity is lacking from human antibody responses to the gB/MF59 vaccine. We showed that CMV seropositive people have high levels of antibodies that neutralize viral entry into epithelial cells. The gB/MF59 vaccine, however, fell far short of inducing the levels of epithelial entry neutralizing activities found in seropositive sera. We hypothesize that a vaccine capable of eliciting both gB- specific antibodies and antibodies that neutralize epithelial entry will have a significantly higher efficacy in preventing primary infection than that of the gB/MF59 vaccine. The epitopes that are targeted by the epithelial entry-specific neutralizing antibodies found in human CMV seropositive sera are unknown, but a strong case can be made for epitopes within a recently discovered virion glycoprotein complex consisting of gH, gL, UL128, UL130, and UL131 (gH/gL/UL128-131). This complex is essential for efficient viral entry into epithelial and endothelial cells but dispensable for entry into fibroblasts, and animal antibodies raised to epitopes within UL128, UL130, or UL131 specifically neutralize epithelial and/or endothelial entry. We therefore propose that vaccine strategies to elicit antibodies to epitopes within the gH/gL/UL128-131 complex will result in potent epithelial entry neutralizing responses. We have partnered with Vical Inc. to develop and evaluate immunization strategies that incorporate DNA expression vectors, recombinant subunit proteins/complexes, and inactivated virions. We will evaluate these approaches for their relative ability to elicit potent epithelial entry neutralizing responses in small animals. Optimal candidate strategies will be further evaluated for compatibility with established gB-based vaccine approaches. The results of the proposed studies will produce candidates ready for clinical development. Human cytomegalovirus (CMV) is the major infectious cause of birth defects in the United States. We recently found that natural infection by CMV elicits neutralizing antibodies that block viral entry into both fibroblasts and epithelial cells, while current candidate vaccines are deficient in eliciting antibodies that block epithelial cell entry. The aim of this project is to develop vaccine candidates that in animals elicit antibodies that block CMV entry into both cells types. Such candidates are anticipated to have improved efficacy over existing candidate vaccines and will be suitable for subsequent clinical development.