Browsing by Author "Alabbad, Aljawharah"

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Developing Novel Protein-Based Strategies for Combating Infectious Disease Transmission
(University of Manchester, 2024) Alabbad, Aljawharah; Derrick, Jeremy
Recent advances in synthetic biology has substantially expedited the study of proteins to enable better study and control of pathogens. This thesis focuses on Toxin-antitoxin (TA) towards developing novel antimicrobial treatments and subsequently investigates virus-like particles (VLPs) as platforms for developing novel vaccines. TA modules are involved in antibiotic response, stress adaptation, bacteriophage resistance, and biofilm formation, making them targets for new antibacterial therapies. The YefM-YoeB TA module in Escherichia coli includes the stable YoeB toxin, a ribosome-dependent endoribonuclease, and the unstable YefM antitoxin, which inhibits YoeB by altering its C-terminal conformation. Extensions of two to four amino acids at YoeB's C-terminusimpair YefM's ability to neutralise YoeB toxicity, while a single amino acid extension does not. These findings highlight the critical role of YoeB's C-terminus in its interaction with YefM and suggest that modifying this interaction could be a novel antibacterial strategy. VLPs are self-assembling bio-nanoparticles that mimic viral nucleocapsid structures, making them ideal for presenting multivalent antigens. Lacking viral genetic material, VLPs are non-infectious and widely used in medical and nanotechnology research. However, genetic fusion or chemical coupling of antigens to VLPs can lead to capsid disassembly, misfolding, and reduced thermal stability, which impedes the generation of a protective immune response. The novel AbBind platform was developed by integrating two Protein A domains into the HBc VLP (HBc PADD). This modification allows the coupling of proteins with Fc domains from IgG antibodies, stabilising conformational epitopes and enabling the attachment of multiple antigens in any combination. Neisseria meningitidis, the cause of meningococcal meningitis and septicaemia, is classified by its polysaccharide capsule, with available effective vaccines for most serogroups. However, developing a vaccine for serogroup B is challenging due to its capsule's similarity to human cell polysaccharides. As a proof of concept, Neisserial adhesion A (NadA) and variants one and three of Factor H binding protein (FHbp) were selected as model antigens and fused to the Fc fragment of mouse IgG2a. A Bio-Layer Interferometry (BLI) assay confirmed the binding of the engineered VLP core particle to the antigen-Fc fusions. Murine immunisation trials comparing Antigen-Fc:HBc PADD complexes to the antigen alone showed that mice vaccinated with the complexes produced robust IgG antibody titres against the respective antigens. Antibodies against antigen-Fc fusions produced in HEK293 cells cross-reacted with E. coli-expressed antigens. Notably, mice immunised with the VLP scaffold and FHbpV1 and FHbpV3 elicited antibodies against both variants. The AbBind platform presenting Fc fusions of NadA or FHbpV3 showed comparable immune responses to bacterial-produced antigens. The findings demonstrate that the engineered AbBind VLP serves as a versatile platform, enhancing IgG antibody production and increasing valency to facilitate strain coverage, thereby significantly expediting vaccine development.
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