Immunization with theHaemophilus ducreyiHemoglobin 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 antibodydependent 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.

Identification and analysis of three virulence-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens

“…For example, immunoproteomic analysis of the whole cell proteins of Riemerella antipestifer revealed that TonB-dependent receptor was among the immunogenic outer membrane proteins (Q. , and a study with a Znuptake Tdr, ZnuD, of Neisseria meningitidis showed that it could induce bactericidal antibodies upon immunization of mice (Stork et al 2010). Similarly, Tdr with vaccine potentials have been found in Haemophilus ducreyi and, as reported by us, P. fluorescens (Afonina et al 2006, Fusco et al 2010, Leduc et al 2011, Y. Hu et al 2012. In this study, we found that, when used as subunit vaccines, the recombinant proteins rTdr1, rTdr2, and rTdr3 elicited moderate but significant protections that differed in magnitude, with the highest RPS being effected by rTdr3.…”

An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding

“…Whole-cell immunoprecipitation was performed as previously described (11), with pig anti-nHgbA antibodies that recognize surface-exposed epitopes of nHgbA (1). H. ducreyi strains were grown on chocolate agar plates with streptomycin (50 g/ml).…”

“…Elimination of chancroid, through either vaccination or diagnosis and antibiotic treatment of core groups, could potentially slow human immunodeficiency virus transmission where both diseases are endemic (26,27). HgbA is a highly effective vaccine in the swine model of chancroid infection (1), and it is conceivable that an HgbA vaccine might be used for the control of chancroid in humans.…”

Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor

“…Bovine Hgb was coupled to Affigel-10 (Bio-Rad, Hercules, CA) as per the manufacturer's instructions. The Hgbagarose was washed and mock eluted before use with elution buffer as previously described (11). Bacteria expressing mutant hgbA in FX547 grown in low-heme GC broth (3 ml at an OD 600 of 1.0) were pelleted, Growth phenotype assay in Hgb-only media.…”

“…HgbA is one of three TonB-dependent transporters (TBDT) present in the genome of prototypical strain 35000HP and the only TBDT absolutely necessary to establish infection in the human experimental model of chancroid (9,10). Immunization with native HgbA (nHgbA) prevents infection in the experimental swine model of chancroid (11,12). Furthermore, passive immunization with HgbA antisera protects against an experimental challenge, suggesting that vaccine protection is mediated by the humoral immune response elicited to nHgbA (13).…”