Novel recombinant anti-mycolic acid immunoglobulin tools for improved understanding and management of tuberculosis

Abstract

Tuberculosis (TB) is an infectious disease that is caused by the Mycobacterium tuberculosis (M.tb) species of bacteria. Despite this being discovered for over 100 years the disease continues to cause epidemics worldwide. The diagnosis of TB is a challenging aspect, with the current tests exhibiting many problems including: long time period between testing and accurate diagnosis, not enough sensitivity, not always accurate, and, in some cases, expensive. Thus novel biomarkers are urgently needed to aid in the management of the disease. Mycolic acids (MAs) are complex lipid molecules that are found in the cell wall of mycobacteria and in particular in M.tb. The fact that these lipids are species-specific makes them a key component for understanding the M.tb organism. While the isolation of these compounds from the organism can be a complex and costly process, the production of stereo-controlled, chemically synthetic tuberculous, mycobacterial MAs can now be conducted in the laboratory. In TB patient sera, antibody immune activity to MA is always accompanied by antibody immune activity to cholesterol. Investigations into the cholesteroid nature of MA have been underway for many years. Researchers have tried to find the MA subclass responsible for this characteristic of the lipids. In this study, we apply recombinant monoclonal antibodies to MA to correlate the cholesteroid functionality of mycolic acids to their structure by means of crossreactivity of antigen recognition. This thesis reports the generation of three different chickenderived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but differently to the three individual classes. The first antibody recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains – CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, probably due to the enhanced binding cooperativity, aiding in the sensitivity. Cholesterol was only recognized by the two monoclonal antibodies that also were capable of recognizing trans-keto-mycolic acids, suggesting that the cholesteroid nature of mycolic acids is determined by the conformation of MA that is induced by this stereo-isomer of keto-mycolic acids. The 2017 World Health Organisation ministerial conference held in Moscow emphasized the dire need for faster acting drugs and technologies that quickly diagnose TB and determine the degree of drug resistance to improve the management of TB worldwide. It is anticipated that the gallibody tools developed here will find application in quick, affordable TB screening tests aimed at regular monitoring of people at risk of infection as well as to determine how TB patients respond to their anti-TB therapies.