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ABSTRACTHaemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyifrom its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyiand partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyiinfection, possibly by preventing acquisition of the essential nutrient heme.

ABSTRACTHaemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyiclass I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAIdispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyistrain 35000HP expressing either class I or class II HgbA (35000HPhgbAIand 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyistrain 35000HPhgbAIbut not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAIIfrom nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAIbut not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAIfrom both studies bound whole cells of 35000HPhgbAIbetter than 35000HPhgbAIIand partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

ABSTRACTHgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbAby deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in transin host strain H. ducreyiFX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.