RESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis and presents with diverse clinical manifestations. Naturally occurring infection occurs following contamination of cuts or skin abrasions, or ingestion of contaminated water, and occasionally through inhalational of infected soil or water particles. The influence of the route of disease acquisition on the efficacy of medical countermeasures has not been explored in humans or in appropriate animal models. The efficacy of co-trimoxazole against melioidosis acquired by different routes of exposure was assessed in postexposure prophylaxis (PEP) and treatment studies in marmoset models of melioidosis. Following challenge with B. pseudomallei by the inhalational, subcutaneous, or ingestion routes of administration, animals were given co-trimoxazole at 12 hourly intervals for 14 days, starting either 6 h postchallenge or at the onset of fever. Animals were then observed for 28 days. All animals that received antibiotic 6 h postchallenge survived the duration of dosing. All animals that received antibiotics at the onset of fever completed the treatment, but 10%, 57%, and 60% of those with ingestion, subcutaneous, and inhalation challenge relapsed, respectively. Bacteriological and histological differences were observed between placebo-control animals and those that relapsed. Immunological profiles indicate difference between animals given placebo and those that relapsed or survived the duration of the study. A broad T-cell activation was observed in animals that survived. Overall, these data suggest the efficacy of co-trimoxazole, as measured in the incidence of relapse, differs depending on the disease-acquisition route. Therefore, there are implications in treating this disease in regions of endemicity.
Asunto(s)
Burkholderia pseudomallei , Melioidosis , Humanos , Animales , Melioidosis/tratamiento farmacológico , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Antibacterianos/uso terapéutico , Agua , Modelos Animales de EnfermedadRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, which is a Gram negative, facultative intracellular bacterium. Disease is prevalent in SE Asia and in northern Australia, as well as in other tropical and subtropical regions. Recently, there is an increasing awareness of the importance of bacterial ingestion as a potential route of infection, particularly in cases of unexplained origin of the disease. The marmoset is a New World Monkey (NWM) species that is being developed as an alternative NHP model to complement the more traditionally used Old World Monkeys (OWM). Models have been developed for the traditional routes of disease acquisition, subcutaneous and inhalational. This manuscript details the development and characterisation of an ingestion model of melioidosis. Dose-ranging study assessed the lethality of B. pseudomallei and disease progression was assessed by euthanizing animals at predetermined time points, 12, 36, 48 and 54 hours post-challenge. Challenge doses of greater than 6.2 x 106 cfu resulted in an acute, lethal, febrile disease. Following challenge the lung was the first organ, outside of the gastrointestinal tract, to become colonised. Enteritis (duodenitis, ileitis and/or jejunitis) was observed in sections of the small intestine from animals that succumbed to disease. However, the most severe pathological features were observed in the mesenteric lymph nodes from these animals. These findings are consistent with lymphatic draining as route of dissemination.
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Burkholderia pseudomallei/fisiología , Sistema Linfático/microbiología , Melioidosis/patología , Animales , Burkholderia pseudomallei/patogenicidad , Callithrix , Modelos Animales de Enfermedad , Enteritis/microbiología , Femenino , Pulmón/microbiología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Masculino , Melioidosis/microbiologíaRESUMEN
Common marmosets (Callithrix jacchus) were shown to be susceptible to inhalational infection with Coxiella burnetii, in a dose-dependent manner, producing a disease similar to human Q fever, characterized by a resolving febrile response. Illness was also associated with weight loss, liver enzyme dysfunction, characteristic cellular activation, circulating INF-γ and bacteraemia. Viable C. burnetii was recovered from various tissues during disease and from 75% of the animal's lungs on 28 days post challenge, when there were no overt clinical features of disease but there was histological evidence of macrophage and lymphocyte infiltration into the lung resulting in granulomatous alveolitis. Taken together, these features of disease progression, physiology and bacterial spread appear to be consistent with human disease and therefore the common marmoset can be considered as a suitable model for studies on the pathogenesis or the development of medical counter measures of inhalational Q fever.
Asunto(s)
Callithrix , Fiebre Q , Administración por Inhalación , Animales , Modelos Animales de Enfermedad , Humanos , Fiebre Q/diagnóstico , BazoRESUMEN
Historically, disease progression in animal models of Q fever has been carried out using PCR to monitor the presence of Coxiella burnetii DNA in the host. However, the colonization and dissemination of other bacterial infections in animal models are tracked using viable counts, enabling an accurate assessment of viable bacterial load within tissues. Following recent advances in the culture methods, it has become possible to do the same with C. burnetii. Here we compare and contrast the different information gained by using PCR or viable counts to study this disease. Viable bacteria were cleared from organs much faster than previously reported when assessed by bacterial DNA, but weight loss and clinical signs improved while animals were still heavily infected.
RESUMEN
We have resequenced the genomes of four Burkholderia pseudomallei K96243 laboratory cultures and compared them to the reported genome sequence that was published in 2004. Compared with the reference genome, these laboratory cultures harbored up to 42 single-nucleotide variants and up to 11 indels, including a 31.7-kb deletion in one culture.
RESUMEN
There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.
Asunto(s)
Vacunas Bacterianas/inmunología , Francisella tularensis/fisiología , Glicoconjugados/inmunología , Hexosiltransferasas/inmunología , Tularemia/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Inhalación , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Pseudomonas aeruginosa/metabolismo , Ratas , Ratas Endogámicas F344 , VacunaciónRESUMEN
BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.
Asunto(s)
Proteínas Bacterianas/genética , Biología Computacional/métodos , Genes Esenciales , Peste/microbiología , Yersinia pestis/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Mutación , Yersinia pestis/crecimiento & desarrollo , Yersinia pestis/metabolismoRESUMEN
Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.
Asunto(s)
Proteínas Bacterianas/genética , Genes Esenciales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Peste/microbiología , Yersinia pestis/crecimiento & desarrollo , Yersinia pestis/genética , Biología Computacional , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Fenotipo , VirulenciaRESUMEN
Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1â3 linked 2-O-acetyl-6-deoxy-ß-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.
Asunto(s)
Glicoconjugados/química , Glicoconjugados/inmunología , Manosa/química , Melioidosis/prevención & control , Oligosacáridos/química , Animales , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Burkholderia pseudomallei/fisiología , Femenino , Ratones , Ratones Endogámicos BALB C , Oligosacáridos/síntesis químicaRESUMEN
Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia pseudomallei/patogenicidad , Sistemas de Secreción Tipo V/genética , Virulencia/genética , Inmunidad Adaptativa , Animales , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Línea Celular , Modelos Animales de Enfermedad , Humanos , Inmunidad Celular , Lectinas/metabolismo , Melioidosis/metabolismo , Melioidosis/patología , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Mutagénesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Suero/química , Sistemas de Secreción Tipo V/metabolismoRESUMEN
The marmoset model of melioidosis was used to explore whether there was any difference in the disease presentation and/or the lesion formation following inhalational challenge with one of four strains of Burkholderia pseudomallei (K96243, 1026b, HBPUB10303a and HBPUB10134a). Marmosets were challenged with a range of bacterial doses and bacterial load, histological and physiological features were determined temporally following lethal disease. Melioidosis presented as an acute, febrile disease with bacteraemia, bacterial dissemination, necrotizing hepatitis, splenitis and pneumonia which was independent of the challenge strain. Generally, there were no major differences in the manifestation of melioidosis following challenge by the different strains of B. pseudomallei; however, there were some differences in the time to death and the severity of the pathological features. The pathological features observed in the liver and spleen of animals challenged with B. pseudomallei strain 1026b were statistically less severe (P < 0.05) and less frequent. However, more severe foci of disease were evident in the lungs of animals challenged with strain 1026b. In all cases, the lesions developed from small areas of bacteria-infected macrophages surrounded by non-infected neutrophils into large lesions with both immune cell types infected. The marmoset model was a useful tool enabling the distinction of subtle difference in the pathological response to B. pseudomallei.
Asunto(s)
Burkholderia pseudomallei/patogenicidad , Exposición por Inhalación , Hígado/patología , Pulmón/patología , Melioidosis/patología , Bazo/patología , Aerosoles , Animales , Carga Bacteriana , Burkholderia pseudomallei/clasificación , Callithrix , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Interacciones Huésped-Patógeno , Hígado/microbiología , Pulmón/microbiología , Macrófagos/microbiología , Macrófagos/patología , Masculino , Melioidosis/microbiología , Neutrófilos/patología , Índice de Severidad de la Enfermedad , Bazo/microbiología , Factores de TiempoRESUMEN
Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10(8) cfu Burkholderia pseudomallei within 22-85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10(2) cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10(2) cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.
Asunto(s)
Burkholderia mallei , Burkholderia pseudomallei , Muermo/microbiología , Muermo/patología , Melioidosis/microbiología , Melioidosis/patología , Animales , Antígenos Bacterianos , Carga Bacteriana , Callithrix , Modelos Animales de Enfermedad , Femenino , Muermo/mortalidad , Inyecciones Subcutáneas , Masculino , Melioidosis/mortalidad , Índice de Severidad de la EnfermedadRESUMEN
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Inmunoconjugados/administración & dosificación , Lipopolisacáridos/inmunología , Melioidosis/prevención & control , Fragmentos de Péptidos/inmunología , Toxina Tetánica/inmunología , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/química , Femenino , Inmunidad Humoral/efectos de los fármacos , Inmunización , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoglobulina G/biosíntesis , Lipopolisacáridos/química , Melioidosis/inmunología , Melioidosis/microbiología , Melioidosis/mortalidad , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Toxina Tetánica/química , Balance Th1 - Th2 , Vacunas ConjugadasRESUMEN
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use of B. pseudomallei as well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge with B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Cápsulas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0079461.].
RESUMEN
The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.
Asunto(s)
Adhesinas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/fisiología , Multimerización de Proteína , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Adhesión Bacteriana/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/virología , Melioidosis/microbiología , Ratones , Mutación , Fenotipo , Estructura Cuaternaria de ProteínaRESUMEN
Burkholderia pseudomallei is a dangerous human pathogen. Phosphoantigens specifically the target primate specific γ9(+)δ2(+) T cells subset and some have been developed as potential immunotherapeutics. Previously, we demonstrated that, when stimulated with the phosphoantigen CHDMAPP, γ9(+)δ2(+) T cells aid in the killing of intracellular B. pseudomallei bacteria. Moreover, we found that common marmoset (Callithrix Jacchus) γ9(+) T cells increase in frequency and respond to the phosphoantigen CHDMAPP and/or B. pseudomallei, in combination with IL-2, in a similar manner to human γ9(+)δ2(+) T cells. Here we evaluate the efficacy of the phosphoantigen CHDMAPP, in combination with IL-2, as a therapy against B. pseudomallei infection, in vivo. We found that the previous studies predicted the in vivo responsiveness of γ9(+) T cells to the CHDMAPP+IL-2 treatment and significant expansion of the numbers of peripheral and splenic γ9(+) T cells were observed. This effect was similar to those reported in other primate species treated with phosphoantigen. Furthermore, splenocytes were retrieved 7 days post onset of treatment, restimulated with CHDMAPP or heat-killed B. pseudomallei and the cultured γ9(+) T cells demonstrated no reduction in IFN-γ response when CHDMAPP+IL-2 animals were compared to IL-2 only treated animals. Using an established model of B. pseudomallei infection in the marmoset, we assessed the potential for using phosphoantigen as a novel immunotherapy. The CHDMAPP treatment regime had no effect on the progression of respiratory melioidosis and this was despite the presence of elevated numbers of γ9(+) T cells in the spleen, liver and lung and an increased proportion of IFN-γ(+) cells in response to infection. We therefore report that the common marmoset has proven a good model for studying the effect in vivo of γ9(+) T cell stimulation; however, γ9(+) T cells have little or no effect on the progression of lethal, respiratory B. pseudomallei infection.
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Burkholderia pseudomallei/inmunología , Callithrix , Inmunoterapia/métodos , Melioidosis/veterinaria , Enfermedades de los Monos/tratamiento farmacológico , Enfermedades de los Monos/inmunología , Linfocitos T/metabolismo , Animales , Citometría de Flujo , Interferón gamma/metabolismo , Interleucina-2/inmunología , Melioidosis/tratamiento farmacológico , Melioidosis/inmunología , Fosfoproteínas/inmunología , Fosfoproteínas/farmacología , Fosfoproteínas/uso terapéuticoRESUMEN
Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l(-1) of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.
Asunto(s)
ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas , Exotoxinas/inmunología , Francisella tularensis/inmunología , Antígenos O/inmunología , Tularemia/prevención & control , Vacunas Conjugadas , Factores de Virulencia/inmunología , ADP Ribosa Transferasas/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/metabolismo , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Campylobacter jejuni/enzimología , Escherichia coli/metabolismo , Exotoxinas/metabolismo , Femenino , Francisella tularensis/metabolismo , Glicosilación , Hexosiltransferasas/metabolismo , Inmunoglobulina G/sangre , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Antígenos O/metabolismo , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Tecnología Farmacéutica , Tularemia/inmunología , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule.
Asunto(s)
Vacunas Bacterianas/inmunología , Burkholderia/inmunología , Melioidosis/prevención & control , Animales , Cápsulas Bacterianas/inmunología , Cápsulas Bacterianas/metabolismo , Vacunas Bacterianas/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
Burkholderia pseudomallei is the causative agent of the disease melioidosis, which is prevalent in tropical countries and is intractable to a number of antibiotics. In this study, the antibiotic co-trimoxazole (trimethoprim/sulfamethoxazole) was assessed for the post-exposure prophylaxis of experimental infection in mice with B. pseudomallei and its close phylogenetic relative Burkholderia mallei, the causative agent of glanders. Co-trimoxazole was effective against an inhalational infection with B. pseudomallei or B. mallei. However, oral co-trimoxazole delivered twice daily did not eradicate infection when administered from 6h post exposure for 14 days or 21 days, since infected and antibiotic-treated mice succumbed to infection following relapse or immunosuppression. These data highlight the utility of co-trimoxazole for prophylaxis both of B. pseudomallei and B. mallei and the need for new approaches for the treatment of persistent bacterial infection.