Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

Fig. 1. Serological profile of 26 Israeli actively infected tuberculosis patients.

Serum responses by ELISA against a Mtb-H37Rv lysate, b Mtb-CDC1551 lysate, c Mtb-H37Ra lysate and cell-wall fraction, and recombinant proteins d PstS1, e antigen 85b (Ag85b), f CFP10 + ESAT6 complex, g Hbha, and h Malate Synthase. Every symbol represents a single patient, with the legend given on the right side of the figure. ATB patients are in red symbols and negative community controls are in green symbols. A heat map summarizing ELISA signals against the different antigens and lysates is shown in (i). Donor P.4 is marked with a black arrow. All error bars are represented as mean ± SD. All statistics were derived for n = 26 ATB patients versus n = 20 negative community controls, except for (a, b) n = 19 negative community controls. Significance was determined using a two-tailed unpaired Welch’s t test. ns no significance. Data are representative of at least two independent experiments.

Fig. 2. Isolation of anti-PstS1 mAbs from memory B cells of P.4.

a Donor P.4 serum responses taken at three different time points (indicated) to recombinant PstS1 by ELISA. b Isolation of PstS1-specific B cells. Whole-blood-derived lymphocytes were stained for CD19, membrane IgG, and PstS1. A total of 148 positive cells were single-cell sorted. c Pie charts representing the heavy chain sequences that were amplified from the CD19+/IgG+/PstS1+ B cells from P.4. The number in the middle of the pies denotes the total number of sequences, and the colored slices indicate clonally related sequences. Upper panel: all sequences. Lower panel: only the 16 clonally related sequences: clone 1—dark blue, clone 2—purple, clone 3—magenta, clone 4—teal, clone 5—green. d Nucleotide mutations in V_H of the clonal versus nonclonal sequences. Error bars are represented as mean ± SEM. n = 16 clonal Vh sequences and n = 85 nonclonal Vh sequences. e, f are dendrograms (created using Geneious software) of the clonally related sequences, heavy and light chains, respectively. The mAbs selected for expression are colored in red. g–i The binding of nine mAbs by ELISA to recombinant PstS1 (g), Mtb-CDC1551 lysate (h), and Mtb-H37Rv lysate (i). j Comparing binding by ELISA of the mature (mt) and the predicted germline (gl) versions of p4-36 (magenta) and p4–163 (dark blue), to recombinant PstS1, Mtb-CDC1551 lysate, and Mtb-H37Rv lysate. AUC binding score of each antibody was determined as relative to the AUC of the mature antibody, which was normalized to 1 (see also Supplementary Fig. 5). All data are representative of at least two independent experiments (g–j).

Fig. 3. Anti-PstS1 mAbs inhibit Mtb in culture.

a Upper panel: gating strategy of H37Ra-infected macrophages, mCherry positive. PMA-differentiated THP-1 cells preincubated with mAbs p4-163, p4-170, p4-36, p4-141, and the isotype control mAb mGO53. For each treatment n = 90,000 cells were analyzed by flow cytometry. Lower panel: histograms showing the frequencies of intracellular antibody-bound bacteria as detected by anti-human VioBlue antibody staining. For each mAb, the binding histogram is shown in red and compared to the isotype control histogram, which is depicted in a gray overlay. b, c Activity of anti-PstS1 mAbs at indicated concentrations in a human whole blood mycobacterial growth inhibition assay (MGIA) after 96 h of infection with BCG or pathogenic Mtb, respectively. CFU was determined in n = 3 biological repetitions. d Activity of anti-PstS1 mAbs (5 µg/ml) used as IgG1 (named “WT”, full columns) or as N279A Fc variants (named “NA”, empty columns) in MGIA. Black and clear shapes correspond to two independent experiments. CFU was determined in n = 5–6 biological repetitions. e Activity of anti-PstS1 mAbs (5 µg/ml) in MGIA following depletion of CD3+ T cells, CD4+ T cells, CD8+ T cells, blockade of MHC II (anti-HLA), and CD16 or CD32 or both (anti-CD16, anti-CD32, marked with a red rectangle). Black, cayenne, and clear shapes correspond to three representative independent experiments. In each experiment, the data points were compared to average CFU infection in PBS, which was normalized to 1. CFU was determined in n = 4–10 biological repetitions. All error bars are represented as mean ± SD. Significance was determined using a one-tailed unpaired t test (b), two-tailed unpaired t test (c, d) for black shapes (d), or one-way ANOVA with Tukey’s multiple-comparison test (e). All statistical analyses are relative to PBS. ns no significance. Data are representative of at least two independent experiments.

Fig. 4. MAbs p4–170 and p4–36 recognize different epitopes on top of PstS1.

a Top: Ribbon diagrams show the crystal structure of PstS1 in a complex with Fab p4–36 (PDB ID 7DM1). The Fab p4-36 heavy and light chains are colored cornflower cyan and hot pink, respectively. The PstS1 structure is colored blue. The bound phosphate (Pi) is represented as filled balls with oxygen and phosphorus atoms colored red and yellow, respectively. Bottom: an open-up surface-shadowed representation showing the contact interface. Residues involved in hydrogen bonds and Van der Waals contacts are highlighted in orange and yellow, respectively. Positively and negatively charged residues involved in the formation of the salt bridges are highlighted in blue and red, respectively. b Close-up view of the interface between PstS1 and Fab p4-36. The dashed lines indicate hydrogen bonds and salt bridges. c Left: Ribbon diagrams show the crystal structure of PstS1 in complex with Fab p4-170 (PDB ID 7DM2). Right: An open-up surface-shadowed representation showing the contact interface. The color schemes used are the same as in (a). d Close-up view of the interface between PstS1 and Fab p4-170. The dashed lines indicate hydrogen bonds and salt bridges. e Four Clone 1 variants (mAbs p4-9, p4-123, p4-163, and p4-170, dark blue), and mAb p4-36 (magenta), as well as negative control mAb mGO53 (gray), were tested for binding by ELISA to six-point mutant PstS1 proteins. The binding curves of PstS1 mutated in p4-170 contact residues K268E, D279A, and S246G, as well as the PstS1-mutated p4-36 contact residues K136E, D139A, and D140A are shown. The binding curves to wild-type PstS1 are shown in the right panel of the figure.

Fig. 5. Structure modeling of the binding of PstS1 to the PstA–B–C phosphate transporter complex.

a Structural superimposition of the Fab–PstS1 complexes with MetQ of the MetNIQ ABC transporter complex (in gold) (PDB code: 6CVL) suggested a possible binding mode of PstS1 to the PstA–B–C phosphate transporter complex. The bound antibodies show no blockage to the assembly of the PstA–B–C–S complex. b Sequence alignments showing that the MetNIQ complex components share significant sequence similarities to these of the PstA–B–C phosphate transporter complex of Mtb.

Fig. 6. Anti-PstS1 mAbs inhibit Mtb in Balb/c mice.

Activity of anti-PstS1 mAbs: p4-36 (magenta bars), p4-163 (dark-blue bars), as well as the negative control mAb mGO53 (gray bars) at indicated concentrations in Balb/c mice. Mice were injected once intraperitoneally with mAbs at indicated amounts 5 hours prior to aerosol infection with pathogenic Mtb. Mice were euthanized after 2 weeks, and lung bacterial burden measured. Error bars are represented as mean±SD. In each treatment n = 6 mice. Significance was determined using a two-tailed unpaired t test. ns no significance. The results are representative of three independent experiments.