The generation and characterisation of antibody to Mycobacterium tuberculosis

Morley, Gabriella Leah (2023). The generation and characterisation of antibody to Mycobacterium tuberculosis. University of Birmingham. Ph.D.

Morley2023PhD.pdf
Text - Accepted Version
Restricted to Repository staff only until 1 January 2028.
Available under License All rights reserved.
Download (25MB) | Request a copy

Abstract

Tuberculosis (TB) continues to afflict humanity despite the progress that has been made by global control programmes; and the TB problem has become more concerning with the rise of drug resistance and the emergence of strains that are untreatable. Even in drug-sensitive TB the treatment is arduous, costly, and long, presenting a significant burden on patients and healthcare systems. One of the reasons for the substantial chemotherapeutic regime required to treat TB relates to the intrinsic nature of this microorganism. Mtb survives both intracellularly and extracellularly in the human host, with certain subpopulations of bacilli residing in sites which are poorly accessible to immune attack and antibiotic action. These subpopulations of persisters are relatively antibiotic resistant and necessitate the long course of anti-tubercular therapy currently administered. However, poor compliance with this treatment can lead to relapse and the emergence of drug-resistant strains. Novel TB therapeutics are thus urgently required to improve the management of this disease and its poor outcomes. Harnessing the properties of antibodies and combining antibody activity with potent antibiotics could offer an answer. Antibody-antibiotic conjugates (AAC) have been engineered successfully for the management of an infectious agent similar in characteristics to TB. The specificity, bioavailability and prolonged half-life of antibodies allows the conjugated antibiotic to reach the site of infection and have antibacterial activity over a sustained period, to enhance control of persister populations.

The work presented in this thesis describes the approach taken to generate monoclonal antibody candidates which could eventually fulfil a role in an AAC to treat TB. Humoral immunogenicity stimulated by Mtb antigens was initially investigated using human serum from patients with TB disease and latent TB infection (LTBI). A trend emerged whereby patients with active TB produced relatively greater IgG responses than controls, particularly for the IgG1 and IgG3 subclasses. In LTBI, greater IgG responses to Lipoarabinomannan (LAM) and Mtb were associated with lower risk of reactivation to active disease. However, heterogeneity in antibody response was observed between individuals and no specific candidate antigen, as a target for an AAC antibody, became evident. Thus, murine hybridoma were generated using several Mtb antigens and Mtb whole cell. Both IgG1 and IgM monoclonal antibodies (mAbs) were extracted from this process, many of which responded to Ag85 complex and the surface of mycobacteria. The IgG1 mAbs were prime candidates for the creation of an anti-Mtb AAC, therefore the variable regions of these mAbs were sequenced, to preserve the novel antigen-binding site. The functionality of the mAbs was also investigated by producing a colocalisation assay which was analysed through fluorescence microscopy. Harnessing the fluorescence microscopy results, in parallel with phagocytosis flow cytometry data, it was possible to observe the anti-Mtb AAC candidate mAbs during mycobacterial infection. This project therefore delivered novel anti-Mtb mAbs with results which provide a foundation for further work to develop an AAC.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):