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

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.

Despite Being "Known, Highly Promiscuous and Active": Presumed Heterosexuality in the USPHS's STD Inoculation Study, 1946-48.

The Sexually Transmitted Disease Inoculation Study of the United States Public Health Service (USPHS) was a short-term deliberate exposure experiment into the prevention of venereal diseases. Between 1946 and 1948, over 1,300 Guatemalan prisoners, psychiatric patients, soldiers, and sex workers were exposed to syphilis, gonorrhoea, and chancroid. USPHS researchers initially proposed hiring sex workers to "naturally" transmit venereal diseases to male subjects who would then be given various prophylaxes. The researchers were interested in studying the effectiveness of new preventative measures. In other words, the USPHS study was designed to transmit venereal diseases heterosexually from an "infected" female body to the men who, it was assumed, were sexually isolated subjects. However, the researchers did record instances of male-to-male disease transmission among their subject populations, instances that challenged the presumption of heterosexuality on which the study was based.

Putative vaccine candidates and drug targets identified by reverse vaccinology and subtractive genomics approaches to control Haemophilus ducreyi, the causative agent of chancroid.

Chancroid is a sexually transmitted infection (STI) caused by the Gram-negative bacterium Haemophilus ducreyi The control of chancroid is difficult and the only current available treatment is antibiotic therapy; however, antibiotic resistance has been reported in endemic areas. Owing to recent outbreaks of STIs worldwide, it is important to keep searching for new treatment strategies and preventive measures. Here, we applied reverse vaccinology and subtractive genomic approaches for the in silico prediction of potential vaccine and drug targets against 28 strains of H. ducreyi We identified 847 non-host homologous proteins, being 332 exposed/secreted/membrane and 515 cytoplasmic proteins. We also checked their essentiality, functionality and virulence. Altogether, we predicted 13 candidate vaccine targets and three drug targets, where two vaccines (A01_1275, ABC transporter substrate-binding protein; and A01_0690, Probable transmembrane protein) and three drug targets (A01_0698, Purine nucleoside phosphorylase; A01_0702, Transcription termination factor; and A01_0677, Fructose-bisphosphate aldolase class II) are harboured by pathogenicity islands. Finally, we applied a molecular docking approach to analyse each drug target and selected ZINC77257029, ZINC43552589 and ZINC67912117 as promising molecules with favourable interactions with the target active site residues. Altogether, the targets identified here may be used in future strategies to control chancroid worldwide.

2017 European guideline for the management of chancroid.

Chancroid is a sexually acquired infection caused by Haemophilus ducreyi. The infection is characterized by one or more genital ulcers, which are soft and painful, and regional lymphadenitis, which may develop into buboes. The infection may easily be misidentified due to its rare occurrence in Europe and difficulties in detecting the causative pathogen. H. ducreyi is difficult to culture. Nucleic acid amplification tests can demonstrate the bacterium in suspected cases. Antibiotics are usually effective in curing chancroid.

Haemophilus ducreyi: from sexually transmitted infection to skin ulcer pathogen.

PURPOSE OF REVIEW: This article provides an overview of the biology, epidemiology, clinical features, diagnostic tests, and treatment of Haemophilus ducreyi infection, with special reference to the decline of chancroid and the recent emergence of H. ducreyi as a pathogen responsible for chronic limb ulceration clinically similar to yaws. RECENT FINDINGS: Chancroid has declined in importance as a sexually transmitted infection in most countries where it was previously endemic. Chancroid may be caused by either class I or class II H. ducreyi isolates; these two classes diverged from each other approximately 1.95 million years ago. H. ducreyi has recently emerged as a cause of chronic skin ulceration in the Pacific region and Africa. Based on sequencing of whole genomes and defined genetic loci, it appears that the cutaneous H. ducreyi strains diverged from the class I genital strains relatively recently. SUMMARY: H. ducreyi should be considered as a major cause of chronic limb ulceration in both adults and children and appropriate molecular diagnostic assays are required to determine ulcer aetiology. The high prevalence of H. ducreyi-related cutaneous ulceration in yaws-endemic countries has challenged the validity of observational surveys to monitor the effectiveness of the WHO's yaws eradication campaign.

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding.

Oral immunization using HgbA in a recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261 in the temperature-dependent rabbit model.

Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of HIV. An effective vaccine against chancroid has not been developed. In this preliminary study, the gene encoding the H. ducreyi outer membrane hemoglobin receptor HgbA was cloned into the plasmid pTETnir15. The recombinant construct was introduced into the attenuated Salmonella typhimurium SL3261 strain and stable expression was induced in vitro under anaerobic conditions. The vaccine strain was delivered into the temperature-dependent rabbit model of chancroid by intragastric immunization as a single dose, or as three doses administered at two-weekly intervals. No specific antibody to HgbA was elicited after either dose schedule. Although the plasmid vector survived in vivo passage for up to 15 days following single oral challenge, HgbA expression was restricted to plasmid isolates recovered one day after immunization. Rabbits inoculated with the 3-dose booster regimen achieved no protective immunity from homologous challenge. These results emphasize that refinements in plasmid design to enhance a durable heterologous protein expression are necessary for the development of a live oral vaccine against chancroid.

Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from 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. ducreyi and 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. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge.

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 (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI 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 (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but 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-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and 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.

Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin.

Haemophilus ducreyi cytolethal distending toxin (HdCDT) is a tripartite AB toxin, which causes DNA damage in affected cells. We investigated the effects of formaldehyde on the chemical, biological, and immunological properties of the HdCDT complex, which was purified by immobilizing the glutathione S-transferase (GST)-CdtB fusion protein, followed by binding of the CdtA and CdtC recombinant proteins. The HdCDT was treated with increasing concentrations of formaldehyde in the presence of lysine. The treatment of HdCDT at 1 and 0.1 mg protein/ml with 320 and 80 mM of formaldehyde, respectively, resulted in the complete abrogation of cytotoxic activity, loss of DNase activity, and loss of binding capacity to HeLa cells. The toxoid showed protein bands of 75-150 kDa in SDS-PAGE, composed of the three cross-linked CDT components detected by immunoblotting. Three doses of 10 microg protein/mouse of the formaldehyde-treated HdCDT elicited toxin-neutralizing antibodies at titers about 200 times higher than those elicited by the native toxin. The described methodology may be applied to produce immunogenic toxoids from other CDTs, which might be used as candidate components in vaccines against CDT-producing bacteria, including H. ducreyi.

Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis.

OBJECTIVES: Male circumcision is associated with reduced risk of HIV infection. This may be partly because of a protective effect of circumcision on other sexually transmitted infections (STI), especially those causing genital ulcers, but evidence for such protection is unclear. Our objective was to conduct a systematic review and meta-analyses of the associations between male circumcision and infection with herpes simplex virus type 2 (HSV-2), Treponema pallidum, or Haemophilus ducreyi. METHODS: Electronic databases (1950-2004) were searched using keywords and text terms for herpes simplex, syphilis, chancroid, ulcerative sexually transmitted diseases, or their causative agents, in conjunction with terms to identify epidemiological studies. References of key articles were hand searched, and data were extracted using standardised forms. Random effects models were used to summarise relative risk (RR) where appropriate. RESULTS: 26 articles met the inclusion criteria. Most syphilis studies reported a substantially reduced risk among circumcised men (summary RR = 0.67, 95% confidence interval (CI) 0.54 to 0.83), although there was significant between study heterogeneity (p = 0.01). The reduced risk of HSV-2 infection was of borderline statistical significance (summary RR = 0.88, 95% CI 0.77 to 1.01). Circumcised men were at lower risk of chancroid in six of seven studies (individual study RRs: 0.12 to 1.11). CONCLUSIONS: This first systematic review of male circumcision and ulcerative STI strongly indicates that circumcised men are at lower risk of chancroid and syphilis. There is less association with HSV-2. Potential male circumcision interventions to reduce HIV in high risk populations may provide additional benefit by protecting against other STI.

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

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.

Attenuated Salmonella typhimurium SL3261 as a vaccine vector for recombinant antigen in rabbits.

Oral live Salmonella vaccine vectors expressing recombinant guest antigens help stimulate systemic, mucosal, humoral, and cell-mediated immune responses against Salmonella and recombinant antigens. It may be possible to use them effectively against Haemophilus ducreyi, the bacterium that causes chancroid, a sexually transmitted genital ulcer disease. This study aimed to test the feasibility of using oral Salmonella vaccine vectors for the evaluation of chancroid vaccine candidates in the temperature-dependent rabbit model of H. ducreyi infection, an in vivo quantitative virulence assay of inducible immunity. We identified 10(8) to 10(9) CFU to be a safe and immunogenic oral dose range of S. typhimurium SL3261, by monitoring post-administration onset and course of illness and antibody titre by enzyme immunoassay (EIA). We successfully transduced plasmid pTETnir15 into the strain to produce recombinant S. typhimurium SL3261(pTETnir15), successfully expressed tetanus toxin fragment C (TetC) in it, and elicited serum anti-TetC titres of 1:6400 by EIA, 4 weeks after inoculation. The course of experimentally induced H. ducreyi skin lesions in rabbits treated with SL3261(pTETnir15) was similar to that in saline-treated controls. We describe a framework that successfully uses Salmonella as a vector for recombinant control antigen in the rabbit model of H. ducreyi infection, and is suitable for pre-clinical evaluation of Salmonella vector-based H. ducreyi vaccine antigen candidates.

A humoral immune response confers protection against Haemophilus ducreyi infection.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Neither naturally occurring chancroid nor experimental infection with H. ducreyi results in protective immunity. Likewise, a single inoculation of H. ducreyi does not protect pigs against subsequent infection. Accordingly, we used the swine model of chancroid infection to examine the impact of multiple inoculations on a host's immune response. After three successive inoculations with H. ducreyi, pigs developed a modestly protective immune response evidenced by the decreased recovery of viable bacteria from lesions. All lesions biopsied 2 days after the first and second inoculations contained viable H. ducreyi cells, yet only 55% of the lesions biopsied 2 days after the third inoculation did. Nearly 90% of the lesions biopsied 7 days after the first inoculation contained viable H. ducreyi cells, but this percentage dropped to only 16% after the third inoculation. Between the first and third inoculations, the average recovery of CFU from lesions decreased approximately 100-fold. The reduced recovery of bacteria corresponded directly with a fivefold increase in H. ducreyi-specific antibody titers and the emergence of bactericidal activity. These immune sera were protective when administered to naïve pigs prior to challenge with H. ducreyi. These data suggest that pigs mount an effective humoral immune response to H. ducreyi after multiple exposures to the organism.

Haemophilus ducreyi: clinical features, epidemiology, and prospects for disease control.

Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid. Chancroid is common in developing countries and facilitates human immunodeficiency virus transmission. In this review, the clinical features, epidemiology, and prospects for disease control are discussed in the context of experimental and natural infection of humans.

Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model.

The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10(6)CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100microg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.

HIV/STDs in minority women: educational and medical interventions to alter the spread.

HIV is but one form of sexually transmitted disease (STD). Many of the other STDs, especially those that produce ulcerating lesions, such as herpes simplex, syphilis, and chancroid, are associated with increased shedding of HIV if the individual is seropositive, and with increased risk of infection if a seronegative individual with that type of STD has unprotected intercourse with an HIV-positive partner. Thus, control and treatment of other STDs is very important in the management and prevention of the spread of HIV/AIDS.