Stand. of -PA antibody responses. Several immunization combinations were shown to induce high titers of antibody Morusin recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was Morusin notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties. Influenza virus has a number of properties that make it worthy of consideration for use as a viral vector for pathogens that have proven problematic with regard to vaccine development. In addition to the fact that influenza vaccines have an extensive history of safety in the human population and have well-established protocols for large-scale production of both live and inactivated forms, influenza vaccines have been shown to elicit Rabbit Polyclonal to SEPT6 strong mucosal and systemic responses as early as 2 to 6 days postvaccination that encompass both the humoral and the cellular branches of the immune system (4, 7). The majority of neutralizing antibodies generated by influenza vaccines recognize the hemagglutinin (HA) glycoprotein on the viral surface, which has also been reported to have adjuvant-like effects when coadministered with viruslike particles (7, 19). We recently reported that the large segments of foreign antigens can be incorporated as inserts into the HA protein without deleterious effects on HA functions. Specifically, we evaluated chimeric HA proteins containing domains from the protective antigen (PA) of PA and vector constructs. (a) PA structure showing the location and size of the LEF and RBD domains (25). The LEF domain is indicated in yellow, and the RBD is indicated in green. (b) Structural depiction of the influenza virus HA indicating the insertion site of the PA domains. The HA1 subunit is in blue, and the HA2 is in red. (c) Schematic diagram depicting the wild-type HA from A/Aichi/2/68, as well as the organization of the constructs, with respect to location of the PA domains in the primary amino acid sequence. The colors of these domains are coordinated with the structure figures in panels a and b. (d) Schematic diagram depicting the wild-type RV glycoprotein and the organization of the RBD domain in the truncated RV G protein. For a more detailed description refer to the study by Smith et al. (30). inf, influenza virus; SP, signal peptide; TM, transmembrane domain; CD, cytoplasmic domain; rab, RV. Similar studies were carried out utilizing a rabies virus (RV) vector, in which the RV glycoprotein was engineered to express the RBD domain (domain 4) from PA (30). These studies showed that G/PA chimeric proteins were able to incorporate into virus particles and that mice immunized with live or inactivated forms of the RV vector mounted antibody responses recognizing the inserted RBD domain. After a single inoculation with the RV vector, the measured humoral responses were similar to those observed in mice immunized with 150 times that amount of recombinant PA alone. The RV G/PA vector was also shown to stimulate a Th2 type response when given at doses of 50 ng or more (30). Despite the induction of high levels of antibodies specific for PA by both the influenza virus and the RV vectors, we were unable to detect anthrax toxin neutralization activity in the sera of immunized mice following single administrations of antigen or homologous boost regimens. Therefore, the purpose of the present study was to examine the antibody responses induced by both the influenza virus and Morusin RV vectors, as well as a recombinant vaccinia virus (VV) vectors expressing HA/PA chimeric proteins, using alternative prime/boost strategies. Our results show that each vector was capable of inducing specific antibody responses and that a second inoculation with the homologous constructs yielded moderate antibody boosting effects. However, we observed a striking increase in PA-specific antibody titers.