The mycobacterial heparin-binding hemagglutinin is a protective antigen in the mouse aerosol challenge model of tuberculosis

Abstract

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

FIG. 1.

Expression of HBHA by M. tuberculosis as determined by reverse transcription-PCR analyses. (A) Total RNA extracted from the M. tuberculosis Erdman strain (lane 2), CDC1551 strain (lane 3), H37Ra strain (lane 4), or HN878 strain (lane 5) or the M. bovis BCG Pasteur strain (lane 6) or BCG Pasteur strain containing a mutation in the HBHA gene (lane 7) was reverse transcribed by using random primers and then combined with gene-specific primers designed to amplify a ∼600-bp segment of HBHA. Lanes 1 and 10 contained molecular weight markers (100-bp ladder). (B) HBHA was expressed in M. tuberculosis CDC1551 after 30 days of growth in media under normal growth conditions (lane 2), under low-oxygen conditions (lane 3), or under nutrient starvation conditions (lane 4). Similarly, HBHA was expressed in the M. tuberculosis Erdman strain in culture (lane 5), after 6 days of growth in BMMΦ in vitro (lane 6), or after 1 year of growth in the lungs (lane 7) and spleen (lane 8) of a C57BL/6 mouse. Lane 1 contained molecular weight markers (100-bp ladder). The same PCR mixture without reverse transcriptase (lane 8 in panel A and lane 9 in panel B) and the same PCR mixture containing DNA instead of RNA (lane 9 in panel A and lane 10 in panel B) were used as controls, and all cDNA preparations were shown to be free of contaminating DNA as described in Materials and Methods.

FIG. 2.

Antigen preparations of HBHA used as vaccines. (A) HBHA purified by heparin-Sepharose gradient chromatography (lane 1) or by subsequent reverse-phase HPLC (lane 2) was analyzed by SDS-PAGE, and gel lanes were stained with Coomassie blue. (B) Immunoblots containing HBHA purified by heparin-Sepharose gradient chromatography (lane 1) or by subsequent reverse-phase HPLC (lane 2), as well as recombinant His-tagged HBHA purified from M. smegmatis (lane 3), were probed with MAb D2, which recognizes methylated HBHA. The positions of the 28-kDa native HBHA protein and molecular mass standards are indicated on the left.

FIG. 3.

Reduction of M. tuberculosis in mouse tissues following immunization with adjuvant-treated HBHA vaccines. Twenty-eight days following a third vaccination with DDA-MPL adjuvant (DDA-MPL), with HPLC-purified HBHA (nHBHA), or with live BCG vaccine (BCG), C57BL/6 mice were aerogenically challenged with ∼200 CFU of the M. tuberculosis Erdman strain per mouse, and the numbers of viable bacteria in the lungs (A) and spleens (B) were determined 28 days following challenge as previously described (3). (C) By using the immunization and challenge protocol described above, the numbers of M. tuberculosis CFU in the lungs of mice vaccinated with adjuvant only (DDA-MPL), heparin-purified HBHA (nHBHA), recombinant His-tagged HBHA purified from M. smegmatis (rMS-HBHA), or recombinant His-tagged HBHA purified from E. coli (rEC-HBHA) were compared at 28 days following challenge. The asterisks indicate the vaccine groups (each of which contained five mice) in which the bacterial counts were significantly reduced (P < 0.001) compared to the groups that received only adjuvant, as determined by a t test.

FIG. 4.

Histopathological examination of lung tissue (stained with hematoxylin and eosin) from mice 28 days after challenge with M. tuberculosis. Lung sections from mice vaccinated with HPLC-purified HBHA (A), with only the DDA-MPL adjuvant (B), with recombinant His-tagged HBHA purified from M. smegmatis (C), and with BCG vaccine (D) are compared. The arrows indicate areas of dense cellular infiltration and consolidation typical of tuberculosis in mice at 30 days. Magnification, ×100.

FIG. 5.

Recognition of cell surface antigen on the M. tuberculosis Erdman strain by IgG in sera from mice immunized with HPLC-purified HBHA. Paraformaldehyde-treated bacteria were incubated with pooled mouse sera at a dilution of 1:250, followed by fluorescein isothiocyanate-conjugated anti-mouse IgG. Uniform fluorescence was observed for most bacteria (A) found in a colony of M. tuberculosis visualized by phase-contrast microscopy (B). Fluorescence was visualized by using a Nikon Optiphot-2 microscope with a ×100 phase/fluorescent objective; images were photographed with a SPOT RT digital camera, and the composite was produced by using Adobe Photoshop. Magnification, ×1,000.

FIG. 6.

Effect of passive immunization with anti-HBHA monoclonal antibodies on the infection of mouse tissues with M. tuberculosis following aerosol challenge. C57BL/6 mice (five mice in each group) were inoculated intravenously with 250 μg each of IgG purified from MAb D2 and MAb E4 ascites (open bars) or with 250 μg each of IgG (isotype control) purified from MAb BP-G10 and MAb HB-65 ascites (striped bars) ∼24 h prior to aerosol challenge with 100 CFU of the M. tuberculosis Erdman strain. The bacterial burdens in lungs, spleens, and livers were determined by plating 28 days following challenge and compared with the burdens in mouse tissues that received no MAbs (solid bars). Statistical analyses were performed as described in Materials and Methods.

FIG. 7.

Comparison of the IFN-γ responses generated by immunization of mice with HBHA vaccines. (A) Splenocytes from mice immunized three times with the DDA-MPL adjuvant (DDA-MPL), with heparin-purified HBHA (nHBHA), or with recombinant His-tagged HBHA purified from M. smegmatis (rMS-HBHA) were incubated with murine BMMΦ infected with M. bovis BCG at an MOI of 3:1. Supernatants were collected after 72 h, and the IFN-γ concentration was determined by using a cytokine enzyme-linked immunosorbent assay as described in Materials and Methods. (B) Lymphocytes from periaortic and inguinal lymph nodes from mice immunized as described above for panel A were incubated with murine BMMΦ that were primed with heparin-purified HBHA (solid bars), with recombinant His-tagged HBHA purified from M. smegmatis (open bars), or with recombinant His-tagged HBHA purified from E. coli (striped bars). The IFN-γ concentration was measured as described above. The data represent the pooled responses from five mice, and the error bars indicate the standard deviations of the means. Asterisks indicate the vaccine groups that are statistically different from the adjuvant control.