Evaluating Tuberculosis Antibody Profiles

Some previous studies in children have demonstrated improved specificities achieved by combining both protein and glycolipid antigens within serological assays [26 (link)–29 (link)]. Furthermore, several studies have illuminated the potential for heightened sensitivity through the combined analysis of multiple antigen targets, effectively overcoming the interindividual heterogeneity of the human humoral immune response to M. tuberculosis [26 (link)–33 (link)].
We will analyse antibodies concentrations and properties against single protein antigens, single glycolipid antigens [12 (link), 34 (link)–40 (link)], as well as multiple antigens in combination (Table 2). The types of antigens include cell wall fractions, whole cell lysates, and total lipids of M. tuberculosis. The selection of protein antigens is based on results from large protein microarray studies in adults [41 (link)–46 (link)], one large multiplex bead-based study in children [31 (link)], and published and unpublished data from an adult study performed in the U.K (MIMIC study; personal communication M. Tebruegge) [47 ]. In order to enhance specificity, the overlap of the antigen targets for M. tuberculosis with Bacillus Calmette-Guérin (BCG) and other non-tuberculous mycobacteria will be reduced.
Table 2

Key protein, glycolipid, and multiple antigens

Type	Name	Rv number/Full name
Protein	FbpC (Ag85C)	Rv0129c
	PstS3	Rv0928
	PstS1	Rv0934
	PapA4	Rv1528
	GarA	Rv1827
	Apa (Mpt32)	Rv1860
	FbpB (Ag85B)	Rv1886c
	Mpt63	Rv1926c
	Mpt64	Rv1980c
	HspX (Acr)	Rv2031c
	Acg	Rv2032
	Rv2034	Rv2034
	Hrp1	Rv2626c
	EsxO-EsxP	Rv2346-Rv2347
	EspA	Rv3616c
	FbpD (Mpt51)	Rv3803c
	FbpA (Ag85A)	Rv3804c
	EsxB (CFP-10)	Rv3874
	EsxA (ESAT-6)	Rv3875
	EsxA-EsxB (ESAT6-CFP10)	Rv3875-Rv3874
	EspD-EspC	Rv3614-Rv3615
	EspB	RV3881c
	Ag85 complex	Rv3804c-Rv1886c-Rv0129c
Glycolipid	LAM	Lipoarabinomannan
	PDIM	Phthiocerol dimycocerosates
	TDM	Trehalose dimycolates
	TMM	Trehalose monomycolates
	PGL	Phenolic glycolipid
Multiple antigens (H37Rv)	Cell wall fractions	contains proteins and non-protein compounds such as mAGP of M. tuberculosis
	Cell membrane fractions	contains the cytoplasmic membrane and components of the outer lipid layer.
	Whole cell lysates	contains proteins, lipids and carbohydrates present within the M. tuberculosis bacterial cell
	Total hypoxic lipids	containing hypoxic culture M. tuberculosis
	Total normoxic lipids	containing normoxic culture M. tuberculosis

Abbreviations: Acg - alpha-crystallin homolog -coregulated gene; Acr - alpha-crystallin homolog; Ag85 complex - antigen 85 complex; Apa - alanine and proline rich secreted protein; CFP-10 - culture filtrate protein-10; ESAT-6 - early secreted antigenic target-6; EspA - ESX-1 secretion-associated protein A; EsxA/B/C/D/O/P - early secretory antigenic target homolog A/B/C/D/O/P; FbpA/B/C/D - fibronectin binding protein A/B/C/D; GarA - Glycogen accumulation regulator A; Hrp1 - hypoxic response protein 1; HspX - heat shock protein-X; Mpt32/51/63/64 - Proteins purified from Mycobacterium tuberculosis 32/51/63/64; PapA4 - polyketide synthase (PKS) associated protein; PstS1/3 - periplasmic phosphate-binding lipoprotein S1/3

Together with targeted M. tuberculosis antigens, this study will evaluate the following distinct properties of the antibodies: isotypes and their subclasses, FcR binding profiles, and antibody glycosylation patterns (refer to Fig. 1). The rational for this is to obtain further information about the immune response to the antigen. TB disease results from a combination of the mycobacteria infecting and the resulting pathologic immune response. Therefore, antibody concentrations may only reflect on exposure, timepoint, and burden of mycobacteria, whereas additional properties such as FcR may reflect on the fact if the immune response producing tissue damage and pathology or not. This is shown in studies in children with TB disease that have demonstrated the potential enhancement of serological assay sensitivity through the integration of diverse antibody isotypes [48 (link)–50 (link)]. Recent advancements in adult research have indicated that an evaluation of certain antibody properties, such as FcRs binding profiles and glycosylation patterns, could potentially enable the differentiation between TB disease and infection [12 (link), 13 (link)].
Fig. 1

Overview of the antibody properties

Interaction between the surface of M. tuberculosis, binding of the antibody and the recognition of the antibody by an immune cell. Sections A, B, and C detail the different antibody properties: A) antibody isotypes and IgG subclasses B) glycosylation patterns of antibodies, including a core glycan and potential additional sugar residues (1–4) C) activating and inhibiting FcRs with varying affinities for antibody binding

Abbreviations: Mtb -Mycobacterium tuberculosis; FcR -fragmented crystallizable region (Fc) receptor; IgM - immunoglobulin M; IgD - immunoglobulin D, IgG_1 − 4 - immunoglobulin G_1 − 4; IgA - immunoglobulin A, N - N-acetylglucosamine; M - mannose; G - galactose; S - sialic acid; F - fucose

As a quality control and potential normalisation variable, we will measure the total antibody concentration of each isotype and the total antibody concentration binding to distinct FcRs.