A Class I Haemophilus ducreyi Strain Containing a Class II hgbA Allele Is Partially Attenuated in Humans: Implications for HgbA Vaccine Efficacy Trials

Abstract

Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.

Keywords: Haemophilus ducreyi; hemoglobin; vaccines.

FIG 1

Representative Western blots of whole-cell lysates (A) and OMPs (B) prepared from 35000HP (lane 1) and FX548 (lane 2) probed with an antiserum to recombinant HgbA_I and the PAL-specific monoclonal antibody 3B9. The filled arrow indicates intact HgbA, and the open arrow indicates PAL. The relative migration of molecular weight standards is shown to the right of each panel. The ratios of intact HgbA to PAL in whole-cell lysates (C) and OMPs (D) were determined by densitometry; the data represent the means and standard deviations from six Western blots.

FIG 2

Growth kinetics of 35000HP (squares) and FX548 (circles) in brain heart infusion broth supplemented with either bovine heme (A) or human hemoglobin (B) at final concentrations of 5 (open symbols) and 50 (filled symbols) μg/ml. Growth was measured by monitoring CFU over time in three (A) or four (B) independent experiments.