ABSTRACTReplication-competent controlled virus vectors were derived from virulent HSV-1 wildtype strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is co-activated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wildtype virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors - which may be regarded as equivalents of inactivated vaccines - induced detectable protection against lethality caused by wildtype virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and second immunization resulted in complete protection. Once activated vectors also induced far better neutralizing antibody and HSV-1-specific T cells responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus as well as a hemagglutinin-specific T cell response. Single activation of viral replication resulted in a several-fold enhancement of this immune response.IMPORTANCEWe hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, necessary safety should be provided by placing replication of the pathogen under stringent control and of activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above hypothesis as well as suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.