Cytokine profiles of patients infected with Mycobacterium ulcerans and unaffected household contacts

Mycobacterium ulcerans, the cause of Buruli ulcer, is an environmental mycobacterium with a distinct geographic distribution. The reasons why only some individuals who are exposed to M. ulcerans develop ulcers are not known but are likely to reflect individual differences in the immune response to infections with this bacterium. In this study, we investigated cytokine profiles of peripheral blood mononuclear cells (PBMC) from 23 Buruli ulcer patients and 25 household contacts in a region of Australia where Buruli ulcer is endemic. The results showed that following stimulation with M. ulcerans or Mycobacterium bovis BCG, PBMC from Buruli ulcer patients mounted a Th2-type response, which was manifested by the production of mRNA for interleukin 4 (IL-4), IL-5, IL-6, and IL-10, whereas unaffected contacts responded mainly with the Th1 cytokines gamma interferon (IFN-gamma) and IL-12. For example, mRNA for IL-4 was detected in 18 of 23 patients but in only 3 of 25 control subjects (P < 0.0001). By contrast, PBMC from 21 of 25 unaffected individuals produced IFN-gamma compared with 3 of 23 patients (P < 0.0001). IFN-gamma release following stimulation with mycobacteria was markedly reduced in affected subjects. Frequencies of antibodies to M. ulcerans in serum samples from affected and unaffected subjects were similar, indicating that many of the control subjects had been exposed to this bacterium. Together, these findings suggest that a Th1-type immune response to M. ulcerans may prevent the development of Buruli ulcer in people exposed to M. ulcerans, but a Th-2 response does not.     

Immune response to infection with Mycobacterium ulcerans

Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses to M. ulcerans and M. bovis BCG in patients with M. ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or current M. ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M. ulcerans or M. bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2). Serum from 9 of 11 patients with M. ulcerans disease, but no control subject, contained antibodies to M. ulcerans. The results indicate that patients with M. ulcerans infection mount an immune response to M. ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features of M. ulcerans-induced disease.     

Prophylactic effect of mycobacterium bovis BCG vaccination against osteomyelitis in children with Mycobacterium ulcerans disease (Buruli Ulcer)

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whose Mycobacterium bovis BCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection against M. ulcerans osteomyelitis in children with BU disease.     

Systemic suppression of interferon-gamma responses in Buruli ulcer patients resolves after surgical excision of the lesions caused by the extracellular pathogen Mycobacterium ulcerans

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-gamma (IFN-gamma) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-gamma spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-gamma responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-gamma responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-gamma responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.     

A booster vaccination with Mycobacterium bovis BCG does not increase the protective effect of the vaccine against experimental Mycobacterium ulcerans infection in mice

Buruli ulcer, caused by Mycobacterium ulcerans, is a necrotizing skin disease emerging particularly in West Africa. M. bovis BCG vaccine offers only short-term protection against experimental footpad infection of C57BL/6 mice with M. ulcerans, and the duration of this protection cannot be prolonged by a booster vaccination.     

Cytokine response to antigen stimulation of whole blood from patients with Mycobacterium ulcerans disease compared to that from patients with tuberculosis

Mycobacterium ulcerans disease (Buruli ulcer) is a skin-ulcerating infection common in some parts of the tropics. We have investigated cytokine secretion after stimulation of whole blood from Buruli ulcer (BU) patients in a region of endemicity in Ghana with M. ulcerans sonicate or culture filtrate antigens to investigate the development of the response over time and its specificity by comparison with the response to Mycobacterium tuberculosis sonicate in human immunodeficiency virus-negative tuberculosis patients. Significant gamma interferon (IFN-gamma) production in response to whole-blood stimulation with M. ulcerans sonicate was detected in patients with ulcers, which was higher than that in patients with nodules but similar to subjects with healed BU. The mean IFN-gamma response in household contacts of BU patients was not significantly different from that in healthy control subjects from an area of nonendemicity. Results in patients with untreated, smear-positive pulmonary tuberculosis and tuberculosis patients on treatment for more than 2 weeks showed that BU patients responded better to M. ulcerans antigens than tuberculosis patients. In contrast, interleukin-10 results were higher in patients with active M. ulcerans disease than in those with healed lesions, but the pattern of response was similar to that seen in tuberculosis. A similar pattern of cytokine secretion was found using M. tuberculosis sonicate as an antigen. Neither of the two culture filtrate antigens of M. ulcerans appeared to be more specific than M. ulcerans sonicate. In the early stages of M. ulcerans disease there was a mixed Th1 and Th2 cytokine response, but the Th1 response emerged as the dominant type.     

Systemic and local interferon-gamma production following Mycobacterium ulcerans infection

Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-gamma levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-gamma levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-gamma levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.     

Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer

Buruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovis BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulcerans homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of M. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.     

Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1-specific immune response

The role of interleukin 10 (IL-10) and gamma interferon (IFN-gamma) on the development of pathology in human Chagas' disease was investigated. Two categories of patients, low and high producers of IFN-gamma, were identified based on the levels of secretion of this cytokine in the supernatant of peripheral blood mononuclear cell (PBMC) cultures. Eighty-three percent of the patients presenting with cardiac disease (CARD) of different degrees and 59% of the patients with the indeterminate form of disease (IND) were identified as high IFN-gamma producers. PBMC from IND patients classified as low IFN-gamma producers secreted significantly higher amounts of IL-10 than did those from other groups. Flow cytometry analysis demonstrated that in PBMC from the IND group, the majority of the IL-10-producing cells were monocytes (CD14(High+) cells), whereas in the CARD group, the major sources of IFN-gamma were T lymphocytes (CD3(+) CD4(+) cells). These results suggest an association between the production of IFN-gamma by CD3(+) CD4(+) cells and morbidity in Chagas' disease, whereas the production of IL-10 by macrophages/monocytes leads to regulation of the immune response in IND patients. We hypothesize that an exacerbated production of IFN-gamma against Trypanosoma cruzi antigens favors the development of a strong Th1 response in CARD patients, which leads to progression of heart disease.     

Activation of an interleukin-4 mRNA-producing population of peripheral blood mononuclear cells after infection with Mycobacterium bovis or vaccination with killed, but not live, BCG.

This study examines the expression of mRNA for the Th2 cytokine, interleukin-4 (IL-4). Peripheral blood mononuclear cells from deer infected with Mycobacterium bovis or vaccinated with live or killed M. bovis bacillus Calmette-Guérin (BCG) were cultured with mycobacterial antigens. IL-4 mRNA production was assayed using the polymerase chain reaction. Elevated levels of IL-4 mRNA were detected in response to at least one antigen preparation in all animals infected with M. bovis as compared with none of the non-infected control animals. After a primary immunization, elevated levels of IL-4 mRNA were detected in only a proportion of vaccinated animals and this did not correlate with whether the vaccine was live BCG or killed BCG in oil. After boosting, all the animals vaccinated with killed BCG in oil exhibited elevated IL-4 mRNA production whereas none of the animals vaccinated with live BCG showed elevated levels. The data suggest that IL-4 is turned off during the immune response to live BCG, that boosting of low-dose live BCG vaccine may be required to 'imprint' this signal and that this may be important in the development of protective immunity to tuberculosis. Killed BCG in adjuvant is not protective and as with experimental infection with virulent M. bovis it failed to switch off the IL-4 response. IL-4 may be useful as a diagnostic tool and as an in vitro marker of vaccine efficacy.     

Mycobacterium bovis BCG vaccination as prophylaxis against Mycobacterium ulcerans osteomyelitis in Buruli ulcer disease

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. Clinically, the disease presents in the skin as either nonulcerative or ulcerative forms and often invades bones either subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis represents a severe form of the disease that often requires numerous surgical interventions, even amputations. Surgery is accepted as the present definitive treatment for BU. In the absence of an effective drug treatment, the need for the development of preventive and control strategies becomes paramount. No specific vaccine, however, is presently available for BU. Of 372 consecutive patients in Benin presenting with BU (confirmed by microbiological and histopathological analyses) whose Mycobacterium bovis BCG scar statuses were known, 196 children (<15 years old) and 108 adults had neonatal BCG vaccination scars. Of 196 children with BCG scars, 17 (8.7%) had osteomyelitis, while 7 of 28 children without BCG scars (25.0%) had osteomyelitis. Of 108 adults with BCG scars, 17 (15.7%) had osteomyelitis, while 14 of 40 adults without BCG scars (35.0%) had osteomyelitis. Our results show that effective BCG vaccination at birth provides significant protection against the development of M. ulcerans osteomyelitis in children and adults. Therefore, health authorities should give attention to the enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms of BU and childhood tuberculosis would likewise be improved by this intervention.     

Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.     

Aerosol infection of mice with recombinant BCG secreting murine IFN-gamma partially reconstitutes local protective immunity

To better understand the contribution of interferon-gamma (IFN-gamma) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN-gamma gene disrupted (IFN-gamma-/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN-gamma (BCG-IFN-gamma) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN-gamma-/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN-gamma resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN-gamma by the recombinant BCG did not protect IFN-gamma-/- mice from subsequent challenge with M. tuberculosis. Infection of IFN-gamma-/- peritoneal macrophages in vitro with BCG-IFN-gamma led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN-gamma induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth.     

Bacillus Calmette–Guérin-induced interleukin-12 did not additionally improve clinical and immunologic parameters in asthmatic children treated with sublingual immunotherapy

OBJECTIVE: To evaluate the effect of bacillus Calmette-Guérin (BCG) as an adjuvant to specific sublingual immunotherapy (SLIT) on the cytokine profile of peripheral blood mononuclear cells (PBMCs) and clinical outcome. METHODS: Thirty-two children with asthma and rhinitis allergic to house dust mite (HDM) with negative purified protein derivative (PPD) skin test response were enrolled. After a run-in period of 8 weeks, patients were randomized to receive either SLIT only (n=16) or one dose of BCG immunization before initiation of SLIT (n=16) with a standardized Dermatophagoides pteronyssinus (D. pteronyssinus)+D. farinea 50/50 extract. PPD-negative asthmatics (n=5) allergic to HDM receiving inhaled therapy only were included for comparison of cytokine levels in PBMC cultures. Efficacy was assessed both at the end of run-in and 6 months of treatment periods with criteria including symptom, medication and quality-of-life (QoL) scores, IgE levels, lung function, provocation concentration (PC20), eosinophil count and skin prick tests. IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-gamma levels were determined in antigen specifically and polyclonally stimulated PBMC cultures. RESULTS: Both treatment groups showed significant improvement at the end of 6 months for asthma and rhinitis scores and QoL, number of asthma attacks, amount of beta2-agonists, inhaled and intranasal steroids, blood eosinophil counts and PC20. Interestingly, phytohaemagglutinin (PHA)-stimulated IL-12 and D. pteronyssinus-stimulated IFN-gamma in PBMC were significantly higher in the treatment groups than controls. In addition, IL-12 levels in response to D. pteronyssinus and PHA stimulation were significantly higher in the SLIT+BCG group than the SLIT alone group and controls. CONCLUSION: The present study demonstrates that successful SLIT is parallel to increased IFN-gamma production by PBMC. Although simultaneous BCG vaccination enhanced IL-12 production, it did not additionally improve the clinical outcome.     

Effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of Mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle

Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.     

Cytokine responses to stimulation of whole blood from patients with Buruli ulcer disease in Ghana

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, follows an indolent course of initial progression to ulceration accompanied by extensive tissue damage. It has been suggested that healing disease stages are accompanied by a protective immune response. We hypothesized that interleukin-4 (IL-4)- or IL-10-induced downregulation of Th-1 responses plays a key role in the progression of early BUD and that healing is accompanied by an augmented Th-1 response. Gamma interferon (IFN-gamma), IL-4, and IL-10 responses were measured after in vitro stimulation with phytohemagglutinin (PHA) and tuberculin purified protein derivative (PPD) of whole blood from 39 (23 early- and 16 late-stage) BUD patients and 39 healthy control subjects in Ghana. Additionally, 30 patients with active or treated tuberculosis (TB) serving as PPD-responsive positive controls were studied. Early-stage BUD patients produced significantly lower levels of IFN and IFN-gamma/IL-4 ratios compared to late-stage BUD patients after PHA stimulation. Compared to that of controls, IFN-gamma production after tuberculin stimulation was significantly higher in late-stage but not in early-stage BUD patients (P=0.009). IL-10 and IL-4 levels did not differ between BUD patients and controls, although active TB patients had significantly higher IL-10 production levels than did treated TB patients. Multivariate analysis showed no confounding factors. In conclusion, Th-1 down regulation in early BUD appears to reverse in later stages of BUD, although an association with IL-10 or IL-4 production does not emerge from our data. Here we show differences in Th-1-type cytokine production between early- and late-stage BUD that might reflect an improved immune defense over time.     

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.     

Vaccination of cattle with a CpG oligodeoxynucleotide-formulated mycobacterial protein vaccine and Mycobacterium bovis BCG induces levels of protection against bovine tuberculosis superior to those induced by vaccination with BCG alone

The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies to M. bovis CFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-gamma and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.     

IL-9 is associated with an impaired Th1 immune response in patients with tuberculosis

Although a defective Th1 response has been demonstrated in patients infected with Mycobacterium tuberculosis (Mtb), the mechanisms leading to this defect are not well understood. To study the immune response to Mtb infection, we stimulated PBMC from individuals with latent tuberculosis infection (LTBI) or patients with tuberculosis (TB) with the Mtb specific antigen early secretory antigenic target-6 (ESAT-6). mRNAs for a panel of cytokines were measured using quantitative real-time PCR (qPCR). PBMC from TB patients exhibited low levels of IFN-gamma, IL-12alpha, IL-12beta, and IL-23 mRNA but high levels of IL-9 mRNA. Sera from TB patients blocked the differentiation and function of dendritic cells from TST negative (TST-) donors. Exogenous IL-9 reduced IFN-gamma mRNA expression in PBMC from LTBI by 30% (n=4) and neutralization of IL-9 restored the IFN-gamma mRNA expression in PBMC from TB patients by 66% (n=8). Thus, increased expression of IL-9 may contribute to the development of TB.