Most bacterial surfaces are decorated with capsular polysaccharides (CPSs) that have been used as the main components of glycoconjugate vaccines (composed of CPS covalently coupled to a carrier protein) for the past three decades. Glycoconjugate vaccines have been successful in generating protective immune responses in human populations; however, problems still persist. A major concern is their variable immune response especially in elderly and HIV positive. Additionally, vaccines are generated with poorly controlled conjugation chemistries, which result in inadequately characterized, heterogeneous, and variably immunogenic vaccines. Mechanisms by which the immune system interacts with the components of the vaccine need to be understood to design knowledge-based, new-generation glycoconjugate vaccines. We utilize an in depth structural and biochemical approach to delineate the mechanisms of carbohydrate epitopes binding with MHCII. We employ a model bacterial polysaccharide antigen, CPS of type 3 Streptococcus pneumoniae (Pn3), conjugated to carrier protein or peptide. In addition to utilizing Pn3 polysaccharide as a vaccine target, we investigate a bacterial glycosyl hydrolase that degrades to be used as a therapeutic for Pn3. In 1931, a soil-dwelling Bacillus was found to produce an enzyme capable of degrading Pn3P. The expression of this enzyme was inducible in the presence of Pn3P, and the Bacillus strain was able to grow with Pn3P as the carbon source. A few years later, Sickles and Shaw were able to isolate the enzyme producing, Bacillus palustris (renamed Bacillus circulans) strain from soil cultures. We have isolated native Pn3Pd from B. circulans culture supernatant and characterized its activity and degradation products. We recently sequenced the genome of this bacterial strain and identified the gene of the enzyme through mass spectrometry-based proteomics. The recombinantly expressed enzyme purified from BL21 (E. coli) cells displays the same Pn3P depolymerization activity as the natively expressed enzyme. To begin to investigate Pn3Pd as a potential therapeutic enzyme against type III S. pneumoniae we assessed the ability of the enzyme to degrade the capsule on a live virulent type III, WU2 strain. Pn3Pd rapidly strips live WU2 of its capsule and disrupts its ability to form biofilms in vitro. Future experiments will include in vitro lung epithelial cell adherence assays and in vivo colonization and lethal challenge.

Therapeutic and prophylactic approaches for pneumococcal infection

254th National Meeting (2017)

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