Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA protects against infection in the swine model of chancroid

Abstract

The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

FIG. 1.

Purification of HgbA. Various fractions from hemoglobin-agarose affinity purification of HgbA are shown in this Coomassie blue-stained 15% SDS-PAGE gel. Lanes: kDa, kilodalton (molecular marker lane); SM, French-pressed cell starting material; SI, sarcosyl-insoluble fraction; Zw Sol, Zwittergent 3,14-soluble fraction applied to a hemoglobin-agarose column; Zw Ins, Zwittergent 3,14-insoluble fraction; UB, unbound flowthrough fraction not bound to hemoglobin-agarose; HgbA (15 μg by BCA total protein determination); BSA Stds, purified bovine serum albumin used to semiquantitate intact HgbA protein. The purified HgbA shown here was the immunogen for the first vaccine experiment. Similar results were obtained in a second purification, which was used for the second vaccine iteration (not shown).

FIG. 2.

Microscopic examination of lesions. Hematoxylin-eosin staining of skin biopsy sections of pigs 1 week after challenge with live H. ducreyi 35000HP cells. (A) Image of normal pig skin; (B and C) skin cross-sections from HgbA-immunized pigs; (D through F) skin sections of mock-immunized pigs. Magnification values are shown.

FIG. 3.

ELISA. Profiles of specific IgG responses of individual pig sera diluted 1:5,000. The sera from four HgbA-immunized and four mock-immunized pigs were collected at immunization 0 (preimmunization) and 1, 2, and 3 (3 weeks after immunizations 1, 2, and 3, respectively). Sera were tested for IgG reactivity against immobilized HgbA. Bound IgG was detected with alkaline phosphatase-labeled anti-pig IgG. Each sample was run in duplicate on at least three different days. The results were expressed as units of optical density (OD) at 405 nm and represent the mean ± standard deviation (SD).

FIG. 4.

Binding of anti-HgbA to whole H. ducreyi cells. A dilution containing 0.2 μl of each preimmunized (Pre) or postimmunized (Post) HgbA serum was mixed with 2 × 10⁷ CFU of the indicated H. ducreyi strains in a total volume of 100 μl (final dilution of serum, 1:500). After binding for 30 min, bacteria were suctioned and washed and antibody binding was detected with a secondary HRP-conjugated anti-pig IgG antibody followed by chemiluminescence detection. The control anti-DsrA serum was made in a pig by using recombinant DsrA from strain 35000. The serum from each pig was individually tested for binding to whole cells in three separate experiments. The data were combined for each group (HgbA or mock immunized) and expressed as the mean ± SD. There was no significant binding of pre- or postimmunized sera from mock-immunized pigs to whole cells (data not shown). RLU/sec, relative light units/s.

FIG. 5.

Bactericidal killing of H. ducreyi. Survival of H. ducreyi in pooled pig serum in the presence of 25% NHS complement. Target strains are shown on the left. Purified pig IgG was used at 5% concentration relative to normal pig IgG serum levels (final concentration, 0.5 mg/ml). The data are compiled from separate experiments done on at least three different days and represented as the mean ± SD. Statistical difference (P value) was obtained by Student's paired t test.

FIG. 6.

Inhibition of hemoglobin (Hgb) binding by HgbA-immunized pig serum or pig IgG. Purified HgbA was coated onto ELISA plates, the plates were blocked, and the abilities of various antibodies to HgbA to block labeled hemoglobin binding to the receptor were tested. Pre, preimmune; Post, postimmune; Pig IgG, purified pig IgG; anti-rHgbA, rabbit anti-recombinant HgbA (positive control); anti-DsrA I, rabbit anti-recombinant DsrA (negative control). The data are compiled from separate experiments done on at least three different days.