Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis

Abstract

The role of Igs in natural protection against infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB, is controversial. Although passive immunization with mAbs generated against mycobacterial antigens has shown protective efficacy in murine models of infection, studies in B cell-depleted animals only showed modest phenotypes. We do not know if humans make protective antibody responses. Here, we investigated whether healthcare workers in a Beijing TB hospital-who, although exposed to suprainfectious doses of pathogenic Mtb, remain healthy-make antibody responses that are effective in protecting against infection by Mtb. We tested antibodies isolated from 48 healthcare workers and compared these with 12 patients with active TB. We found that antibodies from 7 of 48 healthcare workers but none from active TB patients showed moderate protection against Mtb in an aerosol mouse challenge model. Intriguingly, three of seven healthcare workers who made protective antibody responses had no evidence of prior TB infection by IFN-γ release assay. There was also good correlation between protection observed in vivo and neutralization of Mtb in an in vitro human whole-blood assay. Antibodies mediating protection were directed against the surface of Mtb and depended on both immune complexes and CD4+ T cells for efficacy. Our results indicate that certain individuals make protective antibodies against Mtb and challenge paradigms about the nature of an effective immune response to TB.

Keywords: TB; TB restrictors; antibodies; humoral immunity; immune complex.

Fig. S1.

HCWs highly exposed to TB make detectable antibody responses to Mtb antigens. ELISA of total Ig from active TB patients (active), latently infected HCWs (LTBI), and HCWs with no evidence of latent infection (HEBUI) against soluble lysate from Rv; y axis shows dilution factor (as log₁₀ of dilution) of the most dilute antibody that was reactive above background. **P < 0.01, ***P < 0.001 by Student's t test. ns, not significant.

Fig. 1.

Antibody isolated from some individual HCWs is protective against Mtb infection. (A) Schematic illustrating the mouse protection assay. (1) Mice were injected with 20 mg purified Ig or PBS via the i.p. route. (2) After 5 h, they were infected by aerosol with ∼100–200 cfu Mtb per mouse. (3) After 14 d, mice were killed, and total lung homogenates were plated for cfu enumeration. (4) Plates were read after 3–4 wk (Methods). (B) Mice were infected in four batches (i–iv). Antibodies from three donors (21, 23, and 28) showed protection against TB infection. All three positive donors and two negative donors (24 and 30) were rebled and retested with similar results. Brown represents active TB patients; blue represents LTBI HCWs; and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). *P < 0.05, **P < 0.01 by Student's t test.

Fig. S2.

Antibody persistence in vivo and isotype are not responsible for protection. (A) Antibody subtype titers were measured in vivo from sequential bleeds after injection of 5 mg i.p. as detailed in Methods. Numbers in the legends denote donor numbers. Donor 26 made nonprotective responses, and all three others made protective responses. Blue represents LTBI HCWs, and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). (B) Antibody subtype titers from all 48 HCW donors were measured. Blue represents LTBI HCWs, and red represents HEBUI HCWs. The titers from donors making protective antibody responses are shaded differently from the other donors (green in the LTBI group and brown in the HEBUI group). ns, not significant.

Fig. S3.

Protective antibody requires intact T-cell immunity in the mouse. BALB/c and BALB/c nu/nu mice were injected with antibody and then infected by low-dose aerosol infection. Fourteen days later, mice were killed, and lung homogenates were plated for cfu. Black represents PBS negative control in BALB/c mice; grey represents PBS negative control in nude mice; pale blue represents protective donor 21; and red represents nonprotective donor 26. **P < 0.01 by Student's t test. ns, not significant.

Fig. 2.

Antibody from protective donors requires CD4+ T cells for efficacy in a WBA. (A) Antibody from protective donors but not from nonprotective donors increased growth control of Mtb in whole blood after (i) 48 and (ii) 120 h of incubation. (B) Higher antibody dose (500 µg/mL compared with 50 µg/mL) showed increased protection only from protective donors. (C) Depletion of total T cells using anti-CD3 antibodies (D) abrogates the growth restriction effect of antibodies specifically from protective donors in the WBA. (D) Depletion of CD4+ but not CD8+ T cells or blocking of MHC class II (“anti-HLA”) abrogates the efficacy of antibodies from protective donors in the WBA. Blue represents LTBI HCWs, and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test. ns, not significant.

Fig. S4.

Protective antibodies do not enhance phagocytosis, and depletion against six surface-expressed recombinant antigens does not abrogate protection. (A) Phagocytosis of bacillus Calmette–Guérin by THP-1 cells with or without antibodies from protective and nonprotective donors was measured by flow cytometry. (B) Antibodies from protective and nonprotective donors were depleted against 12 μg total (2 μg each of six) protein as identified in Table S2 before being tested in the WBA. Blue represents LTBI HCWs, and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). *P < 0.05 by Student's t test. ns, not significant.

Fig. 3.

Protective antibodies are directed at the Mtb surface, and immune complexes are critical for efficacy. (A) Depletion of antibody directed against the Mtb surface but not directed against soluble Mtb antigens abrogates their protective effect. Antibody from donors 24 (nonprotective) and 28 (protective) was incubated either (i) with Mtb soluble lysate (Rv lysate) or BSA-coated wells overnight or (ii) against intact Mtb vs. BSA and then used in the WBA. No difference in protective effect was observed when antibody was depleted against soluble Mtb antigens, but protection was abrogated when antibody was depleted against whole Mtb bacteria. (B) Use of Mtb grown in the absence of Tween-80 (−Tw) results in greater Mtb growth restriction when antibodies from protective donors are used in the WBA compared with use of Mtb grown in the presence of Tween-80 (+Tw). (C) Blockade of CD16 and CD32A together but not CD16 alone results in abrogation of Mtb growth restriction from antibodies from protective donors in the WBA. Blue represents LTBI HCWs, and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test. ns, not significant.

Fig. S5.

The WBA is highly correlated with the murine protection assay and can be used to identify additional donors making protective antibodies. Another 24 HCWs (12 LTBI and 12 HEBUI) were consented, and total antibodies were isolated as before and tested in (A) the WBA and (B) the murine protection assay— the WBA experiments were performed in four batches (i–iv) and the mouse challenge experiments in two batches (i and ii). Blue represents LTBI HCWs, and red represents HEBUI HCWs. HCWs making protective responses are represented by paler shades (pale blue/pink). *P < 0.05, **P < 0.01 by Student's t test. ns, not significant.