BACKGROUND: Group B Streptococcus (GBS) is a Gram-positive bacterium that is a common cause of infection in neonates and is the predominant pathogen causing meningitis in infants. Different vaccine formulations have been evaluated in preclinical and/or clinical settings, such as polysaccharide-based vaccines, protein-based, polysaccharide-protein conjugate vaccines, and most recently a Multiple Antigen Presenting System (MAPS) vaccine. MAPS is a vaccine that is being developed in the Malley laboratory at Boston Children’s Hospital that consists of GBS polysaccharides and proteins that are combined using affinity interactions.
The overall aim of this thesis is to contribute towards the development of an effective MAPS vaccine by identifying high capsule-producing GBS isolates, optimizing conditions for polysaccharide purifications, characterizing the immune response to carrier protein derivatives, and creating MAPS complexes with GBS-specific protein carriers.
METHODS: The serotypes of 61 GBS clinical isolates were identified via multiplex PCR. To determine high producing strains, the supernatant polysaccharide concentrations (SPC) for type Ia and III were measured by type 14 pneumococcal and GBS Ia ELISAs, respectively. In addition, type III isolates were grown in different conditions (shaking vs. static, Todd Hewitt Broth vs. Todd Hewitt Broth + 0.5% yeast extracts).
For carrier protein production, genetically conserved alpC and rib were ligated onto a pet21b C terminally tagged rhizavidin (protein made by Rhizobium etli that has a very high affinity for biotin) vector. AlpC was purified and used to immunize rabbits. To determine whether the location of the terminal rhizavidin tag affected the immunogenicity of the proteins, we compared the serum titers of C- and N-terminally tagged proteins by ELISA. Additionally, AlpC was conjugated to purified pneumococcal type I polysaccharide for the creation of MAPS complexes.
RESULTS: We serotyped 61 clinical GBS isolates, of which 18 were type Ia and 33 were type III. The type Ia isolate 21 and type III isolate 25 produced the most polysaccharide relative to the other isolates. The majority of the isolates for type III produced more capsule under shaking conditions and when grown in THY (Todd Hewitt Broth +0.5% yeast extracts).
AlpC and rib were successfully cloned into pet21b CRhiz plasmid and purified. To determine if the position of the rhizavadin tag affected immunogenicity, AlpC-C terminus was sent for immunization onto rabbits. This protein generated similar antibody titers than a N-terminally tagged AlpC.
DISCUSSION: The majority of isolates identified from patients were type Ia and III which corresponds to published data stating that Ia and III are amongst the most common serotypes associated with early onset disease. Production of capsule was greatest in isolate 21 for the Ias and isolate 25 for the III, which may be due to enhanced expression of the cps operon.
The data for serum titers from AlpC revealed no major difference between titers generated by the C-and N-terminally tagged proteins. This suggests that placing the rhizavadin tag at the C-terminus or N-terminus for AlpC does not affect immunogenicity.
CONCLUSION: This work presented in this thesis contributed to the development of an effective GBS MAPS vaccine by finding high producing strains to optimize polysaccharide purifications, and purifying AlpC and immunizing with this carrier protein.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/30870 |
| Date | 03 July 2018 |
| Creators | Alonso, Adrian Miguel |
| Contributors | Franzblau, Carl, Malley, Richard |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.002 seconds