Absynth is developing a novel vaccine against the antimicrobial resistant pathogen Staphylococcusaureus based on essential protein antigens. To develop them as a vaccine for animals or humansthere is need to demonstrate the specificity of vaccine action and design potency assays forvaccine batch release. It is therefore extremely important to determine the roles of these antigens.
An Absynth proprietary antigen, key to its vaccine formulation is DivIB. DivIB is a crucial memberof the cell division machinery in S. aureus. The Foster lab has shown DivIB to be essential fordivision by the creation of a conditional lethal strain. Depletion of DivIB leads to a block of celldivision before complete septum formation, suggesting that the protein is perhaps involved in thetransition between septum initiation and formation of the septal plate. In support of this thetopology of DivIB is such that it has an extracytoplasmic domain able to bind cell wallpeptidoglycan. This domain is also active as a vaccine component.
The project will analyze the role of DivIB in cell division in order to determine its molecularactivity and to therefore design specific assay/s for vaccine batch release validation.
Objectives and experimental approach
- Analysis of the biological activity of DivIB: DivIB is a novel peptidoglycan (PG) bindingprotein domain. The specificity of this binding will be determined using a combination of molecularbiophysics approaches (ITC etc). The PG binding domain will also be mapped by the creation ofspecific deletion mutants and the effect of such deletions on the binding efficacy determined.
Milestone 1 (18 months): DivIB PG binding mapped.
- Vaccination studies and potency validation assays: The identification of importantsubdomain(s) of DivIB will provide a platform for the development of Absynth’s productspecification package, i.e., assays for vaccine lot-to-lot consistency for batch release. Theidentification of subdomains will lead the student to evaluate those responsible for vaccine2mediated protection via vaccine animal model trials. Protective efficacy would be based on theevaluation of microbiological, immunological and physiological markers.
Milestone 2 (24 months) Functional assay development and DivIBsubfragment-based vaccinogenrefinement
- DivIB may be part of the apparatus involved in localization of the division machinery. Thusthe effect of DivIB depletion on other members of the machinery will be determined. We havedeveloped our own Stochastic Optical Reconstruction Microscope (STORM), allowing molecularresolution of the division machinery. The location of DivIB during the cell cycle, as well as that ofkey division components (FtsZ, EzrAetc) in a DivIB deficient strain will be determined. Superresolution microscopy will be used to evaluate if DivIB recognizes the peptidoglycan throughspecific architectural structures in it. A primary output of the cell division process is a new septalcell wall. The Foster lab has developed specific fluorescent D-amino acid labeling for STORM inS. aureus. The role of DivIB in PG biosynthesis will be determined to map the point during divisionat which it is required and provde target validation for vaccination studies.
Milestone 3 (42 months) Role of DivIB in cell division established.