We have noticed anecdotally that, in several of our vaccination studies, certain of our negative animal control groups often showed protective efficacy above what we would have expected to observe. In particular, negative control groups of animals that contained an immunostimulatory adjuvant molecule (IAM) without the target antigen provided modest protection against challenge with lethal doses of viral (e.g., influenza virus) or fungal (e.g., Aspergillus fumigatus) pathogens. Further, the protective effect appeared to be enhanced by the presence of the cholesterol maleimide (CMI) moiety that is incorporated into the VesiVax(r) CALV (conjugatable adjuvant lipid vesicles) formulation to facilitate conjugation of the target antigen to the liposomes; in this test group, the target antigen has not been conjugated to the VesiVax(r) CALVs. There also seemed to be a time dependence for the protective effect, i.e., we observed that challenge with the pathogen one week after the last vaccination resulted in more effective protection than challenge three weeks or more after the last vaccination. Based on these observations, we designed and executed a more controlled vaccination study to examine whether or not the protective effect was real and to demonstrate that the addition of the CMI ligand enhances this effect. The most thoroughly studied IAM we have tested in the VesiVax(r) CALV platform is monophosphoryl lipid A (MPL), a Toll-like Receptor (TLR) 4 agonist. We thus used MPL as a model IAM in our preliminary studies. Our results suggest that, indeed, significant protection from pathogen challenge can be achieved without having a target antigen present in the vaccine and that the CMI does improve the protective immune response. We hypothesize that the CMI is facilitating the stimulation of protective immunity via the innate immune response. Thus, we propose in this SBIR Phase I application, to examine the potential commercial utility of this discovery in greater detail with a goal of demonstrating that unconjugated VesiVax(r) CALVs have the potential to be used as a universal vaccine against different pathogens. To maximize the impact of these studies, we will test this concept in an outbred mouse model of lethal influenza challenge.