[Show abstract][Hide abstract]ABSTRACT: Paediatric tuberculosis (TB) is a key indicator for recent transmission and presents a reservoir for the disease. We describe trends in epidemiology, microbiological characteristics and treatment outcome in Denmark between 2000 and 2009. Data was retrieved from the national TB surveillance system and the International Reference Laboratory of Mycobacteriology.In total, 323 TB cases were reported in children below the age of 15 years, accounting for 7.6% of all notified cases in Denmark. The overall incidence rate of childhood TB declined from 4.1 per 100,000 to 1.9 per 100,000 in the study period. Immigrant children comprised 79.6% of all cases, with the highest incidence rate of 94.1 per 100,000 children in 2001. In contrast to immigrant children, the majority of Danish children were younger than 5 years and with known exposure to TB. Pulmonary TB was the commonest presentation. Only half of the cases were culture-confirmed. We observed an overall decreasing trend in the children to adult notification ratio, but a slight increase in the ratio when calculated specifically for ethnic Danes.Childhood TB needs continuous attention with special focus on risk groups. Emphasis on improving early TB case detection, contact tracing and further implementation of preventive treatment is necessary.
Full-text · Article · Aug 2013 · European Respiratory Journal
[Show abstract][Hide abstract]ABSTRACT: CD4 TB10.4 responses and Ag85B CD8 responses in lungs 6 weeks post infection. Mice were immunized with recombinant vaccines as described in figure 2. Six weeks after the final vaccinations mice were subjected to aerosol infection with M.tb, and intracellular cytokine analysis was performed by flow cytometry on lung cells six weeks after infection. The samples analyzed are the same as in figure 3. Cells were stimulated with TB10.4 for CD4 response analysis (A) and Ag85B for CD8 response analysis (B). Bars represent percentages of CD4 (A) or CD8 T cells (B) from individual lungs from 3 mice per group producing any combination of IFN-γ, TNF-α or IL-2 as indicated below the graphs. Background levels obtained in media-stimulated samples have been deducted. Pie charts represent the relative distribution of CD4 or CD8 T cells subsets producing different cytokine combinations as shown in the histograms.
[Show abstract][Hide abstract]ABSTRACT: Here we report for the first time on the immunogenicity and protective efficacy of a vaccine strategy involving the adjuvanted fusion protein "H28" (consisting of Ag85B-TB10.4-Rv2660c) and Modified Vaccinia Virus Ankara expressing H28. We show that a heterologous prime-boost regimen involving priming with H28 in a Th1 adjuvant followed by boosting with H28 expressed by MVA (H28/MVA28) induced the highest percentage of IFN-γ expressing T cells, the highest production of IFN-γ per single cell and the highest induction of CD8 T cells compared to either of the vaccines given alone. In contrast, in mice vaccinated with adjuvanted recombinant H28 alone (H28/H28) we observed the highest production of IL-2 per single cell and the highest frequency of antigen specific TNF-α/IL-2 expressing CD4 T cells pre and post infection. Interestingly, TNF-α/IL-2 expressing central memory-like CD4 T cells showed a significant positive correlation with protection at week 6 post infection, whereas the opposite was observed for post infection CD4 T cells producing only IFN-γ. Moreover, as a BCG booster vaccine in a clinically relevant non-human primate TB model, the H28/H28 vaccine strategy induced a slightly more prominent reduction of clinical disease and pathology for up to one year post infection compared to H28/MVA28. Taken together, our data showed that the adjuvanted subunit and MVA strategies led to different T cell subset combinations pre and post infection and that TNF-α/IL-2 double producing but not IFN-γ single producing CD4 T cell subsets correlated with protection in the mouse TB model. Moreover, our data demonstrated that the H28 vaccine antigen was able to induce strong protection in both a mouse and a non-human primate TB model.
[Show abstract][Hide abstract]ABSTRACT: Meta analysis of four independent experiments. Mice were immunized in two weeks intervals with recombinant vaccines. Six weeks after the final vaccination mice were subjected to aerosol infection with M.tb., and the bacterial burden was determined 6 weeks after infection. Bars represent mean and SEM of estimated log10 protection values from N = 22–23 mice per group from a total of four individual experiments. Log10 protection values were obtained by deducting log10 CFU values for each individual mouse from the mean of the none-vaccinated group in each separate experiment. *p<0.05, **p<0.01, ***p<0.001 as indicated in the graph using one-way ANOVA and Newman-Keul’s post test for multiple comparisons.
[Show abstract][Hide abstract]ABSTRACT: Correlation between CFU and pre- and post infection CD8 responses. A, the splenic total IFN-γ CD8 T cell response obtained using ICS and flow cytometry one week after immunizations shown in figure 2 (TB10.4-stimulation) was correlated to the corresponding mean log10 CFU value obtained in the lungs six weeks after infection and shown in figure 3 within each group. Points represent mean percentage and SEM (vertical) of CD8 T cells producing IFN-γ in response to stimulation with indicated antigens from 4 spleens per group plotted on the y-axis and mean and SEM (horizontal) log10 CFU values of individual mice on the x-axis. Each point represents one group as indicated in the graph. B, the pulmonary total IFN-γ CD8 T cell response obtained using ICS and flow cytometry six weeks after infection shown in figure 3 (TB10.4-stimulation) was correlated to the corresponding mean log10 CFU value shown in figure 3A within each group. Points represent individual mean and SEM values as described in A. *p<0.05, **p<0.01, using Pearson’s product-moment correlation coefficient (r) and correlation test.
[Show abstract][Hide abstract]ABSTRACT: Pathology scores at necropsy after challenge. Gross pathology in lungs and other organs. Pathology scores (maximum score is 10) given as individual organ scores of lungs and other organs in the vaccine and control groups after infection with M. tuberculosis.
[Show abstract][Hide abstract]ABSTRACT: Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes", and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level.
Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction. This article is part of a Special Issue entitled: (Trends in Microbial Proteomics).
No preview · Article · Jul 2013 · Journal of proteomics
[Show abstract][Hide abstract]ABSTRACT: Background
M. tuberculosis remains one of the world’s deadliest pathogens in part because of its ability to establish persistent, latent infections, which can later reactivate to cause disease. In regions of the globe where disease is endemic, as much as 50% of the population is thought to be latently infected, complicating diagnosis and tuberculosis control. The tools most commonly used for diagnosis of latent M. tuberculosis infection are the tuberculin skin test and the newer interferon-gamma release assays, both of which rely on an antigen-specific memory response as an indicator of infection. It is clear that the two tests, do not always give concordant results, but the factors leading to this are only partially understood.
In this study we examined 245 healthy school children aged from 12 to 20 years from Addis Ababa, a tuberculosis-endemic region, characterised them with regard to response in the tuberculin skin test and QuantIFERON™ test and assessed factors that might contribute to discordant responses.
Although concordance between the tests was generally fair (90% concordance), there was a subset of children who had a positive QuantIFERON™ result but a negative tuberculin skin test. After analysis of multiple parameters the data suggest that discordance was most strongly associated with the presence of parasites in the stool.
Parasitic gut infections are frequent in most regions where M. tuberculosis is endemic. This study, while preliminary, suggests that the tuberculin skin test should be interpreted with caution where this may be the case.
Full-text · Article · Jun 2013 · BMC Infectious Diseases
[Show abstract][Hide abstract]ABSTRACT: Background
Despite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions.
We screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB).
Though below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.
Full-text · Article · May 2013 · BMC Research Notes
[Show abstract][Hide abstract]ABSTRACT: Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.
[Show abstract][Hide abstract]ABSTRACT: The bacille Calmette-Guérin vaccine provides very efficient protection in standard animal models of Mycobacterium tuberculosis challenge. We show in this article that although bacille Calmette-Guérin controlled M. tuberculosis growth for 7 wk of infection, the protection was gradually lost as the infection entered the chronic phase. The regrowth of M. tuberculosis coincided with an almost complete disappearance of IL-2-producing CD4 T cells. Booster vaccination with a subunit vaccine (Ag85B-ESAT-6+CAF01) expanded IL-2(+) CD4(+) T cell coexpressing either TNF-α or TNF-α/IFN-γ, and the maintenance of this population in the late stage of infection was associated with enhanced control of bacterial growth. The IL-2(+) CD4(+) T cell subsets were KLRG1(-) (nonterminally differentiated), were found to be CD62L(high), and further maintained a pronounced proliferative and cytokine-producing potential in the draining lymph nodes, when the animals were challenged 2 y postvaccination. These results suggest that the CD4(+) KLRG1(-) IL-2-secreting subsets are central memory T cells with the potential to continuously replenish the T cells at the site of infection and prevent attrition and functional exhaustion.
Full-text · Article · May 2013 · The Journal of Immunology
[Show abstract][Hide abstract]ABSTRACT: Tuberculin skin testing is simple and relatively inexpensive, but the specificity of PPD is affected by BCG vaccination.
Determine optimal dose and specificity of recombinant ESAT-6 and CFP-10 (C-Tb) produced in Lactococcus lactis for diagnosis of M. tuberculosis infection.
In a dose finding phase I trial 0.01 or 0.1 µg preserved and unpreserved C-Tb was injected by Mantoux technique in 38 patients with active tuberculosis and induration responses measured. In a phase II specificity trial in 151 uninfected, BCG vaccinated participants 0.1 µg C-Tb was compared to 2 TU PPD.
0.1 µg C-Tb gave a median induration of 15 mm after 2 days. Phenol preservation did not affect the response. The specificity of C-Tb was 99.3% (95% CI 96-100%) regarding indurations ≥5 mm as a positive outcome. This was higher than the specificity of PPD (63% using a cut-off of 5 mm or 92% using a cut-off of 15 mm to adjust for non-specific BCG responses). Local adverse reactions following C-Tb injection included transient itching and discomfort as expected components of the immune response.
C-Tb offers a simple and convenient skin test to diagnose M. tuberculosis infection using a single, universal cut-off unaffected by BCG vaccination.
ClinicalTrials.gov NCT01033929 and NCT01241188.
[Show abstract][Hide abstract]ABSTRACT: We have designed a therapeutic HIV-1 vaccine concept based on peptides together with the adjuvant CAF01. Peptides represented 15 HLA-supertype-restricted subdominant and conserved CD8 T-cell epitopes and 3 CD4 T-helper cell epitopes. In this phase I clinical trial, safety and immunogenicity were assessed in untreated HIV-1 infected individuals in Guinea-Bissau, West Africa. 23 HIV-1-infected individuals were randomized to receive placebo (n=5) or vaccine (n=18). Safety was appraised by clinical follow-up combined with monitoring of biochemistry, haematology, CD4 T-cell counts and HIV-1 viral loads. T-cell immunogenicity was monitored longitudinally by IFNγ ELISPOT. New vaccine-specific T-cell responses were induced in 6/14 vaccinees for whom ELISPOT data were valid. CD4 T-cell counts and viral loads were stable. The study shows that therapeutic immunization is feasible and safe in Guinea-Bissau and that it is possible to redirect T-cell immunity with CAF01-adjuvanted HIV-1 peptide vaccine during untreated HIV-1 infection in some patients. However, relatively few pre-existing and vaccine-induced HIV-1 T-cell responses to CD8 T cell epitopes were detected against HIV-1 using IFNγ ELISPOT in this chronically infected African population.
Full-text · Article · May 2013 · AIDS research and human retroviruses
[Show abstract][Hide abstract]ABSTRACT: Dry powder vaccine formulations are highly attractive due to improved storage stability and the possibility for particle engineering, as compared to liquid formulations. However, a prerequisite for formulating vaccines into dry formulations is that their physicochemical and adjuvant properties remain unchanged upon rehydration. Thus, we have identified and optimized the parameters of importance for the design of a spray dried powder formulation of the cationic liposomal adjuvant formulation 01 (CAF01) composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) via spray drying. The optimal excipient to stabilize CAF01 during spray drying and for the design of nanocomposite microparticles was identified among mannitol, lactose and trehalose. Trehalose and lactose were promising stabilizers with respect to preserving liposome size, as compared to mannitol. Trehalose and lactose were in the glassy state upon co-spray drying with the liposomes, whereas mannitol appeared crystalline, suggesting that the ability of the stabilizer to form a glassy matrix around the liposomes is one of the prerequisites for stabilization. Systematic studies on the effect of process parameters suggested that a fast drying rate is essential to avoid phase separation and lipid accumulation at the surface of the microparticles during spray drying. Finally, immunization studies in mice with CAF01 in combination with the tuberculosis antigen Ag85B-ESAT6-Rv2660c (H56) demonstrated that spray drying of CAF01 with trehalose under optimal processing conditions resulted in the preservation of the adjuvant activity in vivo. These data demonstrate the importance of liposome stabilization via optimization of formulation and processing conditions in the engineering of dry powder liposome formulations.
Full-text · Article · Feb 2013 · Journal of Controlled Release
[Show abstract][Hide abstract]ABSTRACT: Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine induced responses a cocktail of recombinant MAP proteins (MAP0217, MAP1508, MAP3701c, MAP3783 and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different age. Male jersey calves were divided into 3 groups that were vaccinated at 2, 8 or 16 weeks of age and boosted twice at week 4 and 12 relative to the first vaccination. Vaccine induced immune responses, the IFN-γ cytokine secretion and antibody responses were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only minor effect of the second booster. However, significant differences were observed in the immunogenicity of the different proteins and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of the vaccination age as we found no difference in the IgG1 responses when comparing the three vaccination groups. Combined, our results suggest that appropriate age of vaccination should be considered in vaccination protocols and there is a possible interference of vaccine induced immune responses with weaning (week 8).
Full-text · Article · Feb 2013 · Clinical and vaccine Immunology: CVI
[Show abstract][Hide abstract]ABSTRACT: This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.
No preview · Article · Feb 2013 · Current Drug Delivery
[Show abstract][Hide abstract]ABSTRACT: To successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models which mimic latent infection are valuable tools to describe the changes in metabolism which occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional DIGE, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. By label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient starved cultures, and the protein levels of 230 proteins were increased in nutrient starved culture filtrates, while 208 were decreased. By Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient starved cultures supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Besides, decreased abundance of proteins involved in amino acid and protein synthesis was apparent as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional DIGE based proteomics demonstrated an overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.
[Show abstract][Hide abstract]ABSTRACT: Identification of new vaccine adjuvants with immunopotentiating properties commonly involves in vitro evaluations of candidate compounds for their ability to stimulate cells of the immune system. Subsequent elaborate experiments are then performed on only the positive candidates. Here we show how this strategy may miss good candidates due to context-dependent supramolecular characteristics of the candidate compounds, since both a specific molecular structure and the correct presentation of specific parts of the compounds are required for successful stimulation of the cells. Nevertheless, the supramolecular structure is rarely evaluated although changes in this structure may have a drastic impact on the presentation of the compounds to the cells. Synthetic analogues of the mycobacterial cell wall lipid monomycoloyl glycerol (MMG) possess immunopotentiating properties, but their biophysical characteristics are largely unresolved and the structural features determining their immunoactivating properties have been poorly explored. In the present study, we demonstrate that the immunostimulatory activity in vitro correlates with the supramolecular characteristics of the self-assembled MMG nanostructures. Thus, a series of MMG analogues displaying different stereochemistry in the hydrophobic moiety and the polar headgroup were designed and synthesized with different alkyl chain lengths. Stimulation of human monocyte-derived dendritic cells in vitro was clearly dependent on the stereochemistry of the hydrophobic part and on the alkyl chain length but not on the stereochemistry of the hydrophilic glycerol moiety. Small-angle X-ray scattering (SAXS) analysis showed that the immunoactivating analogues self-assembled into lamellar phases whereas the biologically inert analogues adopted inverse hexagonal phases. Langmuir monolayers confirmed that analogues with opposite lipid acid configurations displayed different packing modes. These data demonstrate that the biophysical properties and the lipid molecular structure are major determinants for the ability of the MMG analogues to activate antigen-presenting cells. Our findings emphasize the importance of investigating the biophysical and structural properties when assessing the effect of adjuvants in vitro.