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Understanding the Host-Pathogen Interactions During Nasopharyngeal Colonization and Pulmonary Infections to Develop Novel Therapies and Vaccines

Using bacteriophage endolysins as parallel therapeutic and immunizing approaches against pneumococci. An emerging way to address the growing antimicrobial resistance problem is the use of bacteriophages and bacteriophage endolysins. Endolysins are enzymes that can degrade the bacterial peptidoglycan, killing and dispersing biofilm bacteria and its matrix. In preliminary studies, we have purified the endolysins Pal and Cpl-1 from Spn bacteriophages Dp-1 and Cp-1, respectively, as well as a chimeric derivative of Cpl-1 that displays >100-fold increase in antimicrobial activity. We have also shown the ability of these endolysins to lyse planktonic Spn, and importantly we also observed that Cpl-1 and Pal were able to both kill biofilm Spn, as well as disperse the biofilm matrix. In addition, we have shown that upon Spn nasopharyngeal colonization, activation of programmed necrosis, i.e., necroptosis, leads to development of antigen-specific antibodies. Herein, we have proposed to address three overall hypotheses: a) that endolysins are efficient pneumococcal anti-biofilm agents, b) that endolysins can be an effective way to prevent Spn colonization in a serotype (strain) independent manner, and c) that intranasal treatment with endolysins promotes protective immunity, to prevent re-colonization and severe disease. We will use a combination of in vitro and in vivo studies with static and dynamic biofilms, cell culture, mice, biochemistry (to engineer more efficient endolysins), transgenic mice (to define the role of programmed cell death in protective immunity), molecular and immunological techniques and next generation technologies (proteomics) to develop novel endolysin treatments against pneumococcal disease and test their effectiveness in development of long-term serotype independent protective immunity. 

Defining the role of cell death as a modulator of inflammation and immunity. Influenza infection promotes an extremely severe form of secondary bacterial pneumonia, characterized by necrotic lung damage and significantly increased mortality. We aim to identify the molecular mechanisms behind this synergism not only in the pulmonary setting but in extrapulmonary organs. Moreover, we aim to exploit cell death as a way to drive immunotherapies against infectious agents and other diseases. 

Protein-based vaccines. One of the major problems of the great burden of Streptococcus pneumoniae (Spn) infections is the acquisition of antimicrobial resistance and the global spread of resistant clones. These problems get enhanced by the major disadvantages of the current capsular polysaccharide-based vaccines, such as cost, serotype specificity, and the resulting incomplete coverage. This occurs mainly because of disease being caused by serotypes not present in the vaccine (maximum of 23 of the >97 capsule types, only 13 in the conjugate vaccine) and replacement carriage.  Our rationale for this project is that development of a subunit-based vaccine utilizing novel conserved antigenic proteins in conjunction with novel polyphosphazene (PPZ) adjuvants, proven to induce adaptive immunity will deepen the current toolkit to prevent pneumococcal disease without serotype limitations. 


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