RESUMEN
Familial forms of monoclonal gammopathy, defined as multiple myeloma (MM) or Monoclonal Gammopathy of Undetermined Significance (MGUS), are relatively infrequent and most series reported in the literature describe a limited number of families. MM rarely occurs in a familial context. MGUS is observed much more commonly, which can in some cases evolve toward full-blown MM. Although recurrent cytogenetic abnormalities have been described in tumor cells of sporadic cases of MM, the pathogenesis of familial MM remains largely unexplained. In order to identify genetic factors predisposing to familial monoclonal gammopathy, the Intergroupe Francophone du Myélome identified 318 families with at least two confirmed cases of monoclonal gammopathy. There were 169 families with parent/child pairs and 164 families with cases in at least two siblings, compatible with an autosomal transmission. These familial cases were compared with sporadic cases who were matched for age at diagnosis, sex and immunoglobulin isotype, with 10 sporadic cases for each familial case. The gender distribution, age and immunoglobulin subtypes of familial cases were unremarkable in comparison to sporadic cases. With a median follow-up of 7.4 years after diagnosis, the percentage of MGUS cases having evolved to MM was 3%. The median overall survival of the 148 familial MM cases was longer than that of matched sporadic cases, with projected values of 7.6 and 16.1 years in patients older and younger than 65 years, respectively. These data suggest that familial cases of monoclonal gammopathy are similar to sporadic cases in terms of clinical presentation and carry a better prognosis.
Asunto(s)
Gammopatía Monoclonal de Relevancia Indeterminada , Mieloma Múltiple , Paraproteinemias , Niño , Humanos , Gammopatía Monoclonal de Relevancia Indeterminada/diagnóstico , Paraproteinemias/genética , Paraproteinemias/complicaciones , Mieloma Múltiple/patología , Pronóstico , Aberraciones CromosómicasRESUMEN
There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.
Asunto(s)
Protección Cruzada/inmunología , Vacunas Meningococicas/farmacología , Neisseria gonorrhoeae/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Estudios de Casos y Controles , Reacciones Cruzadas/inmunología , Femenino , Gonorrea/inmunología , Humanos , Sueros Inmunes/inmunología , Inmunización/métodos , Masculino , Infecciones Meningocócicas/microbiología , Vacunas Meningococicas/inmunología , Vacunas Meningococicas/metabolismo , Ratones , Ratones Endogámicos BALB C , Neisseria meningitidis/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Estudios Retrospectivos , Serogrupo , Vacunación/métodosRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Chancroide/prevención & control , Vacunas contra Haemophilus/inmunología , Haemophilus ducreyi/inmunología , Adulto , Alelos , Proteínas Bacterianas/administración & dosificación , Proteínas Portadoras/administración & dosificación , Chancroide/inmunología , Chancroide/microbiología , Femenino , Vacunas contra Haemophilus/administración & dosificación , Vacunas contra Haemophilus/genética , Haemophilus ducreyi/genética , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenAsunto(s)
Anticuerpos Monoclonales/administración & dosificación , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/tratamiento farmacológico , Infecciones/tratamiento farmacológico , Anciano , Costo de Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Actividad Bactericida de la Sangre/inmunología , Proteínas Portadoras/metabolismo , Chancroide/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Chancroide/metabolismo , Digoxigenina/metabolismo , Fibrinógeno/metabolismo , Regulación Bacteriana de la Expresión Génica , Haemophilus ducreyi/inmunología , Haemophilus ducreyi/metabolismo , Humanos , Unión Proteica/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Chancroide/prevención & control , Haemophilus ducreyi/patogenicidad , Sueros Inmunes/administración & dosificación , Inmunización Pasiva/métodos , Animales , Anticuerpos Antibacterianos/inmunología , Chancroide/inmunología , Chancroide/patología , Modelos Animales de Enfermedad , Oído/patología , Haemophilus ducreyi/inmunología , Histocitoquímica , Sueros Inmunes/inmunología , Viabilidad Microbiana , Microscopía , Receptores de Superficie Celular/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & controlRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Chancroide/prevención & control , Haemophilus ducreyi/inmunología , Lípido A/análogos & derivados , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Ensayo de Inmunoadsorción Enzimática , Sueros Inmunes/inmunología , Inmunización , Lípido A/administración & dosificación , PorcinosRESUMEN
Resisting the bactericidal activity of naturally occurring antibodies and complement of normal human serum is an important element in the evasion of innate immunity by bacteria. In the gram-negative mucosal pathogen Haemophilus ducreyi, serum resistance is mediated primarily by the trimeric autotransporter DsrA. DsrA also functions as an adhesin for the extracellular matrix proteins fibronectin and vitronectin and mediates attachment of H. ducreyi to keratinocytes. We sought to determine the domain(s) of the 236-residue DsrA protein required for serum resistance and extracellular matrix protein binding. A 140-amino-acid truncated protein containing only the C-terminal portion of the passenger domain and the entire translocator domain of DsrA exhibited binding to fibronectin and vitronectin and conferred serum resistance to an H. ducreyi serum-sensitive strain. A shorter DsrA construct consisting of only 128 amino acids was unable to bind to extracellular matrix proteins but was serum resistant. We concluded that neither fibronectin binding nor vitronectin binding is required for high-level serum resistance in H. ducreyi.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Fibronectinas/metabolismo , Haemophilus ducreyi/metabolismo , Vitronectina/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Matriz Extracelular , Fibronectinas/química , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Vitronectina/químicaRESUMEN
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 FX547 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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Haemophilus ducreyi/metabolismo , Hemoglobinas/metabolismo , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Mapeo Epitopo , Humanos , Modelos Moleculares , Eliminación de Secuencia , PorcinosRESUMEN
The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Fibronectinas/metabolismo , Haemophilus ducreyi/metabolismo , Regulación Bacteriana de la Expresión Génica , Haemophilus ducreyi/clasificación , Haemophilus ducreyi/genética , Mutación , Unión Proteica , Vitronectina/metabolismoRESUMEN
The Ducreyi serum resistance A (DsrA) protein of Haemophilus ducreyi belongs to a large family of multifunctional outer membrane proteins termed trimeric autotransporter adhesins responsible for resistance to the bactericidal activity of human complement (serum resistance), agglutination and adhesion. The ability of DsrA to confer serum resistance and bind extracellular matrix proteins lies in its N-terminal passenger domain. We have previously reported that immunization with a recombinant form of the passenger domain of DsrA, rNT-DsrA, in complete/incomplete Freund's adjuvant, protects against a homologous challenge in swine. We present herein the results of an immunogenicity study in mice aimed at investigating the persistence, type of immune response, and the effect of immunization route and adjuvants on surrogates of protection. Our results indicate that a 20 µg dose of rNT-DsrA administered with alum elicited antisera with comparable bacterial surface reactivity to that obtained with complete/incomplete Freund's adjuvant. At that dose, high titers and bacterial surface reactivity persisted for 211 days after the first immunization. Administration of rNT-DsrA with CpG or imiquimod as adjuvants elicited a humoral response with similar quantity and quality of antibodies (Abs) as seen with Freund's adjuvant. Furthermore, intramuscular administration of rNT-DsrA elicited high-titer Abs with significantly higher reactivity to the bacterial surface than those obtained with subcutaneous immunization. All rNT-DsrA/adjuvant combinations tested, save CpG, elicited a Th2-type response. Taken together, these findings show that a 20 µg dose of rNT-DsrA administered with the adjuvants alum, CpG or imiquimod elicits high-quality Abs with reactivity to the bacterial surface that could protect against an H. ducreyi infection.
Asunto(s)
Adhesinas Bacterianas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Vacunas Bacterianas/química , Haemophilus ducreyi , Inmunidad Humoral , Sistemas de Secreción Tipo V/inmunología , Compuestos de Alumbre/administración & dosificación , Aminoquinolinas/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Islas de CpG , Femenino , Adyuvante de Freund/administración & dosificación , Imiquimod , Sueros Inmunes/inmunología , Cambio de Clase de Inmunoglobulina , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Chancroide/prevención & control , Vacunas contra la Salmonella , Salmonella typhimurium/inmunología , Administración Oral , Animales , Antígenos Bacterianos/genética , Vectores Genéticos , Haemophilus ducreyi/inmunología , Masculino , Plásmidos/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas contra la Salmonella/administración & dosificación , Salmonella typhimurium/genética , Toxina Tetánica/genética , Toxina Tetánica/inmunología , Vacunas Atenuadas/administración & dosificaciónRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales de Origen Murino/química , Chancroide/microbiología , Haemophilus ducreyi/inmunología , Adhesinas Bacterianas/química , Secuencia de Aminoácidos , Animales , Vacunas Bacterianas/química , Chancroide/inmunología , Chancroide/prevención & control , Mapeo Epitopo , Fibrinógeno/química , Fibronectinas/química , Humanos , Hibridomas , Ratones , Datos de Secuencia Molecular , Unión Proteica , Conejos , Vitronectina/químicaRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Chancroide/prevención & control , Haemophilus ducreyi , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Modelos Animales de Enfermedad , Fibrinógeno/metabolismo , Sueros Inmunes/inmunología , Inmunidad Humoral , Inmunoglobulina G/sangre , Proteínas Recombinantes/inmunología , Sus scrofaRESUMEN
Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%-75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%-73.9%) at 18 mutant-strain sites (P = .51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/biosíntesis , Chancroide/microbiología , Haemophilus ducreyi/patogenicidad , Proteínas de la Membrana/biosíntesis , Adulto , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Biopsia , Western Blotting , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Femenino , Haemophilus ducreyi/genética , Haemophilus ducreyi/metabolismo , Humanos , Modelos Logísticos , Masculino , Proteínas de la Membrana/genética , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Chancroide/patología , Haemophilus ducreyi/patogenicidad , Mutación , Adolescente , Adulto , Chancroide/microbiología , Femenino , Haemophilus ducreyi/genética , Humanos , Masculino , Piel/microbiología , Piel/patología , VirulenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Chancroide/prevención & control , Vacunas contra Haemophilus/inmunología , Haemophilus ducreyi/inmunología , Hemoglobinas/metabolismo , Porcinos , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/metabolismo , Proteínas Bacterianas/administración & dosificación , Sitios de Unión de Anticuerpos , Proteínas Portadoras/administración & dosificación , Chancroide/inmunología , Chancroide/microbiología , Chancroide/patología , Modelos Animales de Enfermedad , Vacunas contra Haemophilus/administración & dosificación , Inmunoglobulina G/metabolismo , Inmunoglobulina G/fisiología , Unión Proteica/inmunologíaRESUMEN
The Haemophilus ducreyi outer membrane component DsrA (for ducreyi serum resistance A) is necessary for complete resistance to normal human serum (NHS). When DsrA expression in 19 temporally and geographically diverse clinical isolates of H. ducreyi was examined by Western blotting, 5 of the strains expressed a different immunotype of the DsrA protein (DsrA(II)) than the well-characterized prototypical strain 35000HP (DsrA(I)). The predicted DsrA proteins expressed by the DsrA(II) strains were 100% identical to each other but only 48% identical to that of strain 35000HP. In addition to the DsrA(II) protein, class II strains also expressed variant forms of other outer membrane proteins (OMPs) including NcaA (necessary for collagen adhesion A), DltA (ducreyi lectin A), Hlp (H. ducreyi lipoprotein), major OMP, and/or OmpA2 (for OMP A2) and synthesized a distinct, faster-migrating lipooligosaccharide. Based on these data, strains expressing DsrA(I) were termed class I, and those expressing DsrA(II) were termed class II. Expression of dsrA(II) from strain CIP 542 ATCC in the class I dsrA(I) mutant FX517 (35000HP background), which does not express a DsrA protein, rendered this strain resistant to 50% NHS. This demonstrates that DsrA(II) protein is also critical to serum resistance. Taken together, these results indicate that there are two clonal populations of H. ducreyi. The implications of two classes of H. ducreyi strains differing in important antigenic outer membrane components are discussed.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Haemophilus ducreyi/inmunología , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Actividad Bactericida de la Sangre , Haemophilus ducreyi/clasificación , Humanos , Lipopolisacáridos/análisis , Datos de Secuencia MolecularRESUMEN
Haemophilus ducreyi, the causative agent of chancroid, is highly resistant to the complement-mediated bactericidal activity of normal human serum (NHS). Previously, we identified DsrA (for ducreyi serum resistance A), a major factor required for expression of the serum resistance phenotype in H. ducreyi. We describe here a second outer membrane protein, DltA (for ducreyi lectin A), which also contributes to serum resistance in H. ducreyi. Isogenic dltA mutants, constructed in 35000HP wild-type and FX517 dsrA backgrounds, were more susceptible to the bactericidal effects of NHS than each respective parent, demonstrating the additive effect of the mutations. Furthermore, expression of dltA in H. influenzae strain Rd rendered this highly susceptible strain partially resistant to 5% NHS compared to a vector-control strain. Although primary basic local alignment search tool analysis of the dltA open reading frame revealed no close bacterial homologue, similarity to the beta-chain of the eukaryotic lectin ricin was noted. DltA shares highly conserved structural motifs with the ricin beta chain, such as cysteines and lectin-binding domains. To determine whether dltA was a lectin, ligand blots and affinity chromatography experiments were performed. DltA was affinity purified on immobilized lactose and N-acetylgalactosamine, and N-glycosylated but not glycosidase-treated model glycoproteins bound DltA. These data indicate that DltA is a lectin with specificity for lactose-related carbohydrates (CHO) and is important for H. ducreyi serum resistance.