The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

<div><p>Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In <i>Mycobacterium tuberculosis</i>, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first 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, notably those two that were also able to recognize the <i>trans</i>-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the <i>trans</i>-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.</p></div

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto- MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single- chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first 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, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.S1 Fig. Sequences of gallibody clones produced by antibody engineering. 12) Anti-MA 12, 16) Anti-MA 16, 18) Anti-MA 18, CH1-4 = full length constant region, CH2-4 = truncated constant region, VH = variable heavy chain, VL = variable light chain.S2 Fig. SDS-PAGE analysis illustrating gallibody purification using Ni-NTA affinity columns. A) 12CH1-4, B) 16CH1-4, C) 18CH1-4, D) 12CH2-4, E) 16CH2-4, F) 18CH2-4. Gel lanes 1) Marker, 2) Culture supernatant, 3) Flow through 1, 4) Flow through 2, 5) Washes, 6) Elution 1, 7) Elution 2, 8) Elution 3, 9) Elution 4. Successful purification is demonstrated by the comparable thickness of the 67 kDa band obtained with the culture supernatant (2) and the elutions (6±9).S1 Dataset. Experimental data used for producing Figs 3 and 4.S2 Dataset. Experimental data used for producing Fig 5.S3 Dataset. Experimental data used for producing Fig 6.The Council for Scientific and Industrial Research (CSIR) parliamentary grants (YL) and the National Research Foundation of South Africa for the grants, unique grant numbers: 99386 (HR), 88622, 80577 (YL) and TTK1206281756 (LN).http://www.plosone.orgam2018Biochemistr

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

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.Thesis (PhD)--University of Pretoria, 2018.BiochemistryPhDUnrestricte

Chemically synthetic mycolic acids as vaccine adjuvants

No abstractDissertation (MSc)--University of Pretoria, 2013.BiochemistryMScUnrestricte

Structures of the three major MA classes from <i>Mycobacterium tuberculosis</i>, alpha-MA (1), methoxy-MA (2) and keto-MA (3).

<p>Structures of the three major MA classes from <i>Mycobacterium tuberculosis</i>, alpha-MA (1), methoxy-MA (2) and keto-MA (3).</p

Deduced amino acid sequences of CDR 3 regions for phage displayed monoclonal scFv Abs isolated against MA.

<p>Deduced amino acid sequences of CDR 3 regions for phage displayed monoclonal scFv Abs isolated against MA.</p

Characterization of the binding specificities of MA-specific phage-displayed Abs with ELISA using single stereo-isomers of stereo-controlled synthetic MAs, their 1:1 mixtures with α-MA and natural MA mixture.

<p>In this monoclonal phage ELISA assay, the coating of MA antigens was done by adding 50 μl per well of MA (62.5 μg/ml) dissolved in hexane. Hexane alone was used as a negative control. A) Anti-MA 12, B) Anti-MA 16, C) Anti-MA 18. Error bars = Standard error of mean, n = 8 (biological repeats) t = <i>trans</i>, c = <i>cis</i>. The experiments on all synthetic MAs were performed with the three mAbs in parallel under the same conditions. This result is a representative of more than three biological repeats with six technical repeats for each mAb. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200298#pone.0200298.s003" target="_blank">S1 Dataset</a>.</p

Structures of two types of vectors used for antibody engineering.

<p>A: scFvIgY _(CH1-4) and B: scFvIgY _(CH2-4). Figure adapted from Greunke <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200298#pone.0200298.ref033" target="_blank">33</a>].</p

Evaluation of the selectivity and specificity of MA-reactive fusion-phage clones in ELISA.

<p>Natural MA was used as a positive control, while denatured bovine serum albumin (DBSA), native bovine serum albumin (NBSA) and hexane were used as negative controls. MAs were coated at 0.250 mg/ml while cholesterol was coated at both 0.250 mg/ml and 1 mg/ml. The concentration of bovine serum albumin used was 1 mg/ml. Error bars = Standard error of mean, n = 5 (biological repeats). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200298#pone.0200298.s003" target="_blank">S1 Dataset</a>.</p

Cholesterol cross-reactivity of gallibodies confirmed with ELISA using hexane solvent alone as negative control.

<p>Coating of cholesterol was done at 1 mg/ml in hexane. A) Anti-MA 12, B) Anti-MA 16, C) Anti-MA 18. Error bars = Standard deviation, n = 8 (biological repeats) CH1-4 = full length constant region, CH2-4 = truncated constant region. The results each represent two biological repeats with four technical repeats for each concentration of gallibody. The data were extracted from the same experiment that was performed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200298#pone.0200298.g005" target="_blank">Fig 5</a>, making the absorbance readings from both figures directly comparable. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200298#pone.0200298.s005" target="_blank">S3 Dataset</a>.</p