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Background: The only available tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has variable efficacy. New vaccines are therefore urgently needed. Why BCG fails is incompletely understood, and the tools used for early assessment of new vaccine candidates do not account for BCG variability. Taking correlates of risk of TB disease observed in human studies and back-translating them into mice to create models of BCG variability should allow novel vaccine candidates to be tested early in animal models that are more representative of the human populations most at risk. Furthermore, this could help to elucidate the immunological mechanisms leading to BCG failure. We have chosen the monocyte to lymphocyte (ML) ratio as a correlate of risk of TB disease and have back-translated this into a mouse model. Methods: Four commercially available, inbred mouse strains were chosen. We investigated their baseline ML ratio by flow cytometry; extent of BCG-mediated protection from Mtb infection by experimental challenge; vaccine-induced interferon gamma (IFNγ) response by ELISPOT assay; and tissue distribution of BCG by plating tissue homogenates. Results: The ML ratio varied significantly between A/J, DBA/2, C57Bl/6 and 129S2 mice. A/J mice showed the highest BCG-mediated protection and lowest ML ratio, while 129S2 mice showed the lowest protection and higher ML ratio. We also found that A/J mice had a lower antigen specific IFNγ response than 129S2 mice. BCG tissue distribution appeared higher in A/J mice, although this was not statistically significant. Conclusions: These results suggest that the ML ratio has an impact on BCG-mediated protection in mice, in alignment with observations from clinical studies. A/J and 129S2 mice may therefore be useful models of BCG vaccine variability for early TB vaccine testing. We speculate that failure of BCG to protect from TB disease is linked to poor tissue distribution in a ML high immune environment.
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Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-α, and IL-1ß are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α, and IL-1ß, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1ß and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.
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Citocinas/biosíntesis , Inflamación/inmunología , Interferón Tipo I/biosíntesis , Interleucina-10/análisis , Macrófagos/metabolismo , Animales , Burkholderia pseudomallei/inmunología , Citocinas/inmunología , Interferón Tipo I/inmunología , Interleucina-10/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, which is associated with a range of clinical manifestations, including sepsis and fatal pneumonia and is endemic in Southeast Asia and Northern Australia. Treatment can be challenging and control of infection involves prolonged antibiotic therapy, yet there are no approved vaccines available to prevent infection. Our aim was to develop and assess the potential of a prophylactic vaccine candidate targeted against melioidosis. The identified candidate is the 22kDa outer membrane protein, OmpW. We previously demonstrated that this protein was immunoprotective in mouse models of Burkholderia cepacia complex (Bcc) infections. We cloned Bp_ompW in Escherichia coli, expressed and purified the protein. Endotoxin free protein administered with SAS adjuvant protected Balb/C mice (75% survival) relative to controls (25% survival) (p<0.05). A potent serological response was observed with IgG2a to IgG1 ratio of 6.0. Furthermore C57BL/6 mice were protected for up to 80 days against a lethal dose of B. pseudomallei and surpassed the efficacy of the live attenuated 2D2 positive control. BpompW is homologous across thirteen sequenced B. pseudomallei strains, indicating that it should be broadly protective against B. pseudomallei. In conclusion, we have demonstrated that BpOmpW is able to induce protective immunity against melioidosis and is likely to be an effective vaccine antigen, possibly in combination with other subunit antigens.
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Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Melioidosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Burkholderia pseudomallei , Femenino , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Vacunas de Subunidad/inmunologíaRESUMEN
Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.
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Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/metabolismo , Melioidosis/microbiología , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Factores de Virulencia/genéticaRESUMEN
Melioidosis, a severe human disease caused by the bacterium Burkholderia pseudomallei, has a wide spectrum of clinical manifestations ranging from acute septicemia to chronic localized illness or latent infection. Murine models have been widely used to study the pathogenesis of infection and to evaluate novel therapies or vaccines, but how faithfully they recapitulate the biology of human melioidosis at a molecular level is not known. In this study, mice were intranasally infected with either high or low doses of B. pseudomallei to generate either acute, chronic, or latent infection and host blood and tissue transcriptional profiles were generated. Acute infection was accompanied by a homogeneous signature associated with induction of multiple innate immune response pathways, such as IL-10, TREM1, and IFN signaling, largely found in both blood and tissue. The transcriptional profile in blood reflected the heterogeneity of chronic infection and quantitatively reflected the severity of disease. Genes associated with fibrosis and tissue remodeling, including matrix metalloproteases and collagen, were upregulated in chronically infected mice with severe disease. Transcriptional signatures of both acute and chronic melioidosis revealed upregulation of iNOS in tissue, consistent with the expression of IFN-γ, but also Arginase-1, a functional antagonist of the iNOS pathway, and was confirmed by immunohistochemistry. Comparison of these mouse blood datasets by pathway and modular analysis with the blood transcriptional signature of patients with melioidosis showed that many genes were similarly perturbed, including Arginase-1, IL-10, TREM1, and IFN signaling, revealing the common immune response occurring in both mice and humans.
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Burkholderia pseudomallei/inmunología , Inmunidad Innata/inmunología , Melioidosis/inmunología , Animales , Arginasa/biosíntesis , Arginasa/sangre , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-10/sangre , Interleucina-10/genética , Interleucina-10/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Metaloproteinasa 9 de la Matriz/sangre , Melioidosis/microbiología , Melioidosis/patología , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Receptores Inmunológicos/sangre , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Transducción de Señal/inmunología , Transcriptoma/genética , Receptor Activador Expresado en Células Mieloides 1RESUMEN
There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection.
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Burkholderia pseudomallei/genética , Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , Peroxirredoxinas/genética , Peroxirredoxinas/inmunología , Inmunidad Adaptativa/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Burkholderia pseudomallei/enzimología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Genotipo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Ratones , Ratones TransgénicosRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.
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Proteínas Bacterianas/inmunología , Infecciones por Burkholderia/inmunología , Burkholderia/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Alelos , Animales , Proteínas Bacterianas/química , Vacunas Bacterianas/inmunología , Infecciones por Burkholderia/genética , Burkholderia pseudomallei/inmunología , Reacciones Cruzadas/inmunología , Fibrosis Quística/prevención & control , Epítopos de Linfocito T/química , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunización , Interferón gamma/biosíntesis , Melioidosis/prevención & control , Ratones , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Especificidad del Receptor de Antígeno de Linfocitos T/inmunologíaRESUMEN
Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ΔrelA ΔspoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelA ΔspoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ΔrelA ΔspoT mutant may be a promising live-attenuated vaccine candidate.