The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our IntroductionHeparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by antibodies that recognize multimolecular complexes of platelet factor 4 (PF4), a positively charged platelet protein, and heparin (H), a negatively charged carbohydrate. We, and others, have shown that PF4 and H complexes assemble primarily through nonspecific electrostatic interactions governed by principles of colloidal chemistry. [1][2][3][4][5] In colloidal systems, molecules of opposite charge "aggregate" or grow in size due to effects of charge neutralization. Particle interactions are frequently dependent on stoichiometric ratios of the 2 compounds, with the largest complexes occurring at molar ratios of the compounds leading to charge neutralization. When either compound is in molar excess, charge restabilization occurs and repulsive forces predominate, leading to reduced complex size and/or complex disassembly.Studies to date indicate that PF4/H multimolecular complex formation is central to the pathogenesis of HIT. The characteristic bell-shaped curve seen with HIT antibody binding over a range of H concentrations coincides with H-dependent formation of multimolecular complexes. 2,3 HIT antibody binding, as gauged by serologic assays or functional studies of platelet activation, is optimal when multimolecular complexes form at or near equimolar ratios of PF4:H. However, antibody binding is markedly reduced with increasing H concentrations, a phenomenon that can be directly attributed to loss of complex formation. [2][3][4] Recent studies from our laboratory indicate that similar H-dependent changes affect the immunogenicity of PF4/H complexes in vivo. 5,6 Our studies demonstrate that PF4/H complexes are immunogenic over a certain range of H concentrations associated with multimolecular complex formation and that the immune response is attenuated when PF4 or H is given alone or when H is in molar excess of PF4. 5 H and H-like molecules bind several positively charged proteins in addition to PF4. 7 These H-binding proteins (HBPs) are structurally and functionally diverse, and include, to name a few, nuclear proteins (protamine), enzymes (C1 esterase and lysozyme), adhesion molecules (fibronectin and vitronectin) growth factors (fibroblast growth factor), and lipid-binding proteins (apolipoprotein E and lipoprotein lipase). To date, it appears that a majority of HBP-H interactions a...
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.
Haemophilus 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. ducreyi class I strain 35000HP (nHgbA I ) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbA I dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbA I and 35000HPhgbA II , respectively). Pigs immunized with the nHgbA I /MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbA I but not heterologous strain 35000HPhgbA II , as evidenced by the isolation of only strain 35000HPhgbA II from nHgbA I -immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbA I -immunized animals challenged with strains 35000HPhgbA I but not those challenged with strain 35000HPhgbA II . Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbA I /MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbA I /Freund's vaccine; however, anti-nHgbA I from both studies bound whole cells of 35000HPhgbA I better than 35000HPhgbA II and partially blocked hemoglobin binding to nHgbA I . In conclusion, despite eliciting lower antibody ELISA activity than the nHgbA I /Freund's, the nHgbA I /MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.
HgbA 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 hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi
Previously, we showed that serum resistance in Haemophilus ducreyi type strain 35000HP required expression of the outer membrane protein DsrA because the isogenic dsrA mutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptible dsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing of dsrA mutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5-and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited onto dsrA mutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serumresistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in the dsrA mutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.
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