Project Summary Streptococcus pneumoniae (Spn) remains one of the deadliest infectious agents responsible for more than a million deaths worldwide per year as well as making up a majority of secondary infections in immunocompromised patients. One of the major virulence mechanisms associated with this pathogen is its polysaccharide capsule, which determines successful colonization and infection of the bacterium as well as serving as a defense mechanism against immune-mediated killing of the pathogen. Spn serotype 3 (Spn3) is considered one of the most virulent serotypes of Spn, and despite current multivalent vaccines that include conjugate targets against Spn3, the overall proportion of this serotype in Spn infection cases has actually increased over the last ten years. Furthermore, Spn3 is directly associated with increased antibiotic resistance. Thus, the severity of the impact of Spn3, as well as the difficulties arising in designing therapies against it, cannot be overstated. Pneumozyme (Pn3Pase) is an enzyme product that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn. Our in vitro studies have shown that treating Spn3 with Pneumozyme will render the bacterium more susceptible to killing mechanisms by phagocytic cells. Furthermore, mice that are lethally challenged with Spn3 can be rescued following treatment with Pneumozyme: 100% of mice receiving Pneumozyme treatment survival the challenge whereas 100% of mice that receive inactive enzyme or vehicle die within two days of Spn3 lethal challenge. These results indicate that Pneumozyme has extremely high efficacy in sensitizing Spn3 to host immune mechanisms and that the enzyme has remarkable potential as a therapeutic. As part of Pneumotactix, LLC, our aims for this project are thus: Aim 1: Characterize the pharmacokinetics, potential immunogenicity and cytotoxicity of the enzyme. Aim 2: Elucidate the therapeutic value of the enzyme against Spn3 infection. In our published studies, we have used the native, full-length protein product to degrade the capsule of one virulent strain of Spn3 to great effect, and have further shown that this enzyme product does not induce cellular toxicity against host mammalian cells in both in vitro and in vivo experiments. Our immediate goals for this project are therefore to expand these studies to test pharmacokinetics, assess enzyme efficacy against additional strains of serotype 3 Spn, and perform continued experiments to confirm the therapeutic value of Pneumozyme and concurrent lack of off-target effects upon administration. The long-term goals for Pneumozyme are to establish it as a therapeutic agent by testing its efficacy in higher animal systems (i.e., primates) with significant sample numbers of Spn type 3 clinical isolates, and move towards clinical trials to develop this enzyme as a highly relevant therapeutic against this incredibly virulent pathogen.