Melioidosis is a neglected tropical disease intrinsically resistant to antibiotics, even with prolonged antibiotic therapy there can be a relapse of infection. During infection, Burkholderia pseudomallei exploits an intracellular niche, avoiding extracellular antibiotic therapies. The objective of this research is to target intracellular bacteria with the use of antibodies conjugated to antibiotics. This project will develop and test an antibody-antibiotic conjugate (AAC) to specifically deliver antibiotic to the intracellular site of infection. An AAC has been designed consisting of an anti-Burkholderia antibody conjugated to an antibiotic via a cleavable linker. The mechanism of action relies upon opsonisation of bacteria and uptake of the AAC into phagocytes. Within the phagocyte, the linker between the antibody and antibiotic will be cleaved by cathepsin, therefore releasing the antibiotic in an active form to kill the intracellular bacteria. A macrophage infection assay is the primary method used for analysing the intracellular action of the AAC, together with confocal microscopy, imaging flow cytometry and bacterial colony counts. Monoclonal antibodies have been assessed in vitro for opsonisation ability, resulting in an anti-capsule antibody being selected for incorporation into an AAC. In addition to opsonisation, this antibody also significantly reduced bacterial actin tail formation in vitro. An antibiotic has been successfully chemically conjugated to a monoclonal antibody, via a cathepsin cleavable linker. The AAC generated in this project, demonstrates functionality in vitro as a targeted delivery of antibiotic within macrophage cells. This work represents an initial proof of principle in the development of an AAC as a targeted antibiotic delivery therapy for melioidosis. The targeting of antibiotic delivery is important for anti-microbial resistance concerns, limiting off target effects of the antibiotic. Targeting the intracellular site of infection has the potential to improve current antibiotic therapies for melioidosis, and reduce relapse of infection in infected individuals.
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