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1.  Analysis of the Vaccine Potential of Plasmid DNA Encoding Nine Mycolactone Polyketide Synthase Domains in Mycobacterium ulcerans Infected Mice 
There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.
Author Summary
Buruli ulcer (BU) is an infectious disease, characterized by deep, ulcerating skin lesions, particularly on arms and legs, which are provoked by a toxin. BU is caused by a microbe of the genus that also cause tuberculosis and leprosy. The 33 countries where Buruli ulcer has been detected, especially in West Africa, have mainly tropical and subtropical climates, although the disease is also present in temperate areas of Australia and Japan. There is no effective vaccine against BU and it is still not fully understood which immune defence mechanisms (antibodies and/or T cells) are needed to control the infection. The identification of microbial components that are involved in immune control is an essential step in the development of an effective vaccine. In this paper, we used an experimental mouse model to demonstrate the immunogenicity and the vaccine potential of enzymes involved in the toxin synthesis. Combinations with other vaccine candidates, such as a subunit vaccine against Ag85A targeting cell wall synthesis or with live, attenuated M. bovis BCG or mycolactone negative Mycobacterium ulcerans remain to be tested.
PMCID: PMC3879250  PMID: 24392169
2.  The Immunodominant T-Cell Epitopes of the Mycolyl-Transferases of the Antigen 85 Complex of M. tuberculosis 
The Ag85 complex is a 30–32 kDa family of three proteins (Ag85A, Ag85B, and Ag85C), which all three possess enzymatic mycolyl-transferase activity involved in the coupling of mycolic acids to the arabinogalactan of the cell wall and in the biogenesis of cord factor. By virtue of their strong potential to induce Th1-type immune responses, important for the control of intracellular infections, members of the Ag85 family rank among the most promising TB vaccine candidate antigens. Ag85A and Ag85B, initially purified from Mycobacterium bovis bacillus Calmette–Guérin (BCG)/Mycobacterium tuberculosis culture filtrate respectively, induce strong T-cell proliferation and IFN-γ production in most healthy individuals latently infected with M. tuberculosis and in BCG-vaccinated mice and humans but not in tuberculosis patients. Members of the Ag85 complex are highly conserved in other mycobacterial species. Mice and humans infected with Mycobacterium ulcerans or cattle infected with M. bovis or Mycobacterium avium subsp. paratuberculosis also show strong T-cell responses to this protein family. Using synthetic overlapping peptides, bio-informatic prediction programs and tetramer-binding studies, a number of immunodominant CD4+ and CD8+ T-cell epitopes have been identified in experimental animal models as well as in humans, using proliferation and Th1 cytokine secretion as main read-outs. The results from these studies are summarized in this review.
PMCID: PMC4089088  PMID: 25071781
antigen 85; mycolyl transferase; Th1 helper T-cell; immunodominance; promiscuous epitopes
3.  M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model 
PLoS ONE  2013;8(10):e76442.
The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4+ and particularly CD8+ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG.
PMCID: PMC3798287  PMID: 24146869
4.  Mucosal and Systemic Immune Responses to Mycobacterium tuberculosis Antigen 85A following Its Co-Delivery with CpG, MPLA or LTB to the Lungs in Mice 
PLoS ONE  2013;8(5):e63344.
Pulmonary vaccination is a promising route for immunization against tuberculosis because the lung is the natural site of infection with Mycobacterium tuberculosis. Yet, adjuvants with a suitable safety profile need to be found to enhance mucosal immunity to recombinant antigens. The aim of this study was to evaluate the immunogenicity, the safety and the protective efficacy of a subunit vaccine composed of the immunodominant mycolyl-transferase antigen 85A (Ag85A) and one of three powerful mucosal adjuvants: the oligodeoxynucleotide containing unmethylated cytosine-phosphate-guanine motifs (CpG), the monophosphoryl lipid A of Salmonella minnesota (MPLA) or the B subunit of heat-labile enterotoxin of Escherichia coli (LTB). BALB/c mice were vaccinated in the deep lungs. Our results showed that lung administration of these adjuvants could specifically induce different types of T cell immunity. Both CpG and MPLA induced a Th-1 type immune response with significant antigen-specific IFN-γ production by spleen mononuclear cells in vitro and a tendency of increased IFN-γ in the lungs. Moreover, MPLA triggered a Th-17 response reflected by high IL-17A levels in the spleen and lungs. By contrast, LTB promoted a Th-2 biased immune response, with a production of IL-5 but not IFN-γ by spleen mononuclear cells in vitro. CpG did not induce inflammation in the lungs while LTB and MPLA showed a transient inflammation including a neutrophil influx one day after pulmonary administration. Pulmonary vaccination with Ag85A without or with MPLA or LTB tended to decrease bacterial counts in the spleen and lungs following a virulent challenge with M. tuberculosis H37Rv. In conclusion, CpG and MPLA were found to be potential adjuvants for pulmonary vaccination against tuberculosis, providing Th-1 and Th-17 immune responses and a good safety profile.
PMCID: PMC3651129  PMID: 23675482
5.  Mycobacterium tuberculosis Lacking All Mycolic Acid Cyclopropanation Is Viable but Highly Attenuated and Hyperinflammatory in Mice 
Infection and Immunity  2012;80(6):1958-1968.
Mycolic acids, the major lipid of the Mycobacterium tuberculosis cell wall, are modified by cyclopropane rings, methyl branches, and oxygenation through the action of eight S-adenosylmethionine (SAM)-dependent mycolic acid methyltransferases (MAMTs), encoded at four genetic loci. Mycolic acid modification has been shown to be important for M. tuberculosis pathogenesis, in part through effects on the inflammatory activity of trehalose dimycolate (cord factor). Studies using the MAMT inhibitor dioctylamine have suggested that the MAMT enzyme class is essential for M. tuberculosis viability. However, it is unknown whether a cyclopropane-deficient strain of M. tuberculosis would be viable and what the effect of cyclopropane deficiency on virulence would be. We addressed these questions by creating and characterizing M. tuberculosis strains lacking all functional MAMTs. Our results show that M. tuberculosis is viable either without cyclopropanation or without cyclopropanation and any oxygenated mycolates. Characterization of these strains revealed that MAMTs are required for acid fastness and resistance to detergent stress. Complete lack of cyclopropanation confers severe attenuation during the first week after aerosol infection of the mouse, whereas complete loss of MAMTs confers attenuation in the second week of infection. Characterization of immune responses to the cyclopropane- and MAMT-deficient strains indicated that the net effect of mycolate cyclopropanation is to dampen host immunity. Taken together, our findings establish the immunomodulatory function of the mycolic acid modification pathway in pathogenesis and buttress this enzyme class as an attractive target for antimycobacterial drug development.
PMCID: PMC3370573  PMID: 22431648
6.  Cellular Immunity Confers Transient Protection in Experimental Buruli Ulcer following BCG or Mycolactone-Negative Mycobacterium ulcerans Vaccination 
PLoS ONE  2012;7(3):e33406.
Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy.
Methodology/Principal Findings
In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed.
The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised.
PMCID: PMC3297633  PMID: 22413022
7.  Pertussis Serodiagnosis in Belgium from 1990 to 2009 ▿ 
Diagnosis of pertussis by culture and PCR is most sensitive when performed on nasopharyngeal specimens collected <2 weeks and <3 weeks, respectively, after the onset of clinical disease. Conversely, serological testing allows the diagnosis of patients (mostly adults) with less typical whooping cough symptoms, for whom clinical samples are often collected at later time points. Here, we report on a 20-year serodiagnostic survey of pertussis in Belgium from 1990 to 2009. In total, 13,163 patients were analyzed for Bordetella pertussis-specific antibodies by agglutination, complement fixation, immunofluorescence, and ELISA. The number of positive pertussis cases detected by serodiagnosis ranged between 50 and 150 annually. The mean age of positive cases increased from 9.9 years in 1990 to 33.9 years in 2009. Whereas from 1990 to 2003, children and young adolescents made up the majority of cases, from 2004 onwards, cases were detected in all age groups and the distribution became bimodal, with a first peak at the age of 10 to 20 years and a second at the age of 35 to 50 years. In contrast, patients diagnosed since 2001 by PCR and/or culture were mostly children younger than 1 year of age. Despite extensive childhood vaccination campaigns, whooping cough is still present in Belgium. Our findings confirm the potential role of adults in the continued transmission of pertussis and strongly warrant booster or cocoon vaccinations in older age groups.
PMCID: PMC3122568  PMID: 21346057
8.  Increased Pulmonary Tumor Necrosis Factor Alpha, Interleukin-6 (IL-6), and IL-17A Responses Compensate for Decreased Gamma Interferon Production in Anti-IL-12 Autovaccine-Treated, Mycobacterium bovis BCG-Vaccinated Mice ▿  
Interleukin-12 (IL-12) and IL-23 (which share a p40 subunit) are pivotal cytokines in the generation of protective Th1/Th17-type immune responses upon infection with the intracellular pathogen Mycobacterium tuberculosis. The role of IL-12 and IL-23 in protection conferred by the tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) is, however, less well documented. By using an autovaccine approach, i.e., IL-12p70 cross-linked with ovalbumin and PADRE peptide formulated with the GSK proprietary adjuvant system AS02V, we could specifically neutralize IL-12 while leaving the IL-23 axis intact. Neutralization of IL-12 before M. tuberculosis challenge rendered C57BL/6 mice highly susceptible, resulting in 30-fold-higher CFU in spleen and lungs and accelerated mortality. In contrast, neutralization of IL-12 in BCG-vaccinated mice prior to M. tuberculosis challenge only marginally affected vaccine-mediated protection. Analysis of cytokine production in spleen and lungs 3 weeks post-TB challenge by enzyme-linked immunosorbent assay and functional and flow cytometric assays showed significantly reduced mycobacterium-specific gamma interferon (IFN-γ) responses in M. tuberculosis-infected and BCG-vaccinated mice that had been treated with the autovaccine. Purified protein derivative-induced tumor necrosis factor alpha (TNF-α), IL-6, and IL-17A levels, however, were highest in lungs from BCG-vaccinated/IL-12-neutralized animals, and even unstimulated lung cells from these mice produced significant levels of the three cytokines. Mycobacterium-specific IL-4 and IL-5 production levels were overall very low, but IL-12 neutralization resulted in increased concanavalin A-triggered polyclonal secretion of these Th2-type cytokines. These results suggest that TNF-α, IL-6, and IL-17A may be more important pulmonary effector molecules of BCG-mediated protection than IFN-γ in a context of IL-12 deficiency.
PMCID: PMC3019790  PMID: 21084465
9.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG
10.  Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis▿  
Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.
PMCID: PMC2849333  PMID: 20107000
11.  T Cell Reactivity against Mycolyl Transferase Antigen 85 of M. tuberculosis in HIV-TB Coinfected Subjects and in AIDS Patients Suffering from Tuberculosis and Nontuberculous Mycobacterial Infections 
The mycolyl transferase antigen 85 complex is a major secreted protein family from mycobacterial culture filtrate, demonstrating powerful T cell stimulatory properties in most HIV-negative, tuberculin-positive volunteers with latent M.tuberculosis infection and only weak responses in HIV-negative tuberculosis patients. Here, we have analyzed T cell reactivity against PPD and Ag85 in HIV-infected individuals, without or with clinical symptoms of tuberculosis, and in AIDS patients with disease caused by nontuberculous mycobacteria. Whereas responses to PPD were not significantly different in HIV-negative and HIV-positive tuberculin-positive volunteers, responses to Ag85 were significantly decreased in the HIV-positive (CDC-A and CDC-B) group. Tuberculosis patients demonstrated low T cell reactivity against Ag85, irrespective of HIV infection, and finally AIDS patients suffering from NTM infections were completely nonreactive to Ag85. A one-year follow-up of twelve HIV-positive tuberculin-positive individuals indicated a decreased reactivity against Ag85 in patients developing clinical tuberculosis, highlighting the protective potential of this antigen.
PMCID: PMC2948887  PMID: 20936150
12.  Liver X receptors contribute to the protective immune response against Mycobacterium tuberculosis in mice  
The Journal of Clinical Investigation  2009;119(6):1626-1637.
Liver X receptors (LXRs) are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. However, exactly how LXRs modulate inflammation during infection remains unknown. To explore this, we used a mouse model of Mycobacterium tuberculosis infection. Upon intratracheal infection with M. tuberculosis, LXRs and LXR target genes were induced in CD11c+ lung and alveolar cells. Furthermore, mice deficient in both LXR isoforms, LXRα and LXRβ (Lxra–/–Lxrb–/– mice), were more susceptible to infection, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. Interestingly, mice solely deficient in LXRα, but not those lacking only LXRβ, mirrored the susceptibility of the Lxra–/–Lxrb–/– animals. Lxra–/–Lxrb–/– mice failed to mount an effective early neutrophilic airway response to infection and showed dysregulation of both pro- and antiinflammatory factors in CD11c+ lung cells. T cell responses were strongly affected in Lxra–/–Lxrb–/– mice, showing near-complete abrogation of the infection-induced Th1 function — and even more so Th17 function — in the lungs. Treatment of WT mice with the LXR agonists TO901317 and GW3965 resulted in a 10-fold decrease of the pulmonary bacterial burden and a comparable increase of Th1/Th17 function in the lungs. The dependence of LXR signaling on the neutrophil IL-17 axis represents what we believe to be a novel function for these nuclear receptors in resistance to M. tuberculosis infection and may provide a new target for therapeutics.
PMCID: PMC2689129  PMID: 19436111
13.  PhoP: A Missing Piece in the Intricate Puzzle of Mycobacterium tuberculosis Virulence 
PLoS ONE  2008;3(10):e3496.
Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.
PMCID: PMC2566814  PMID: 18946503
14.  Genetic Resistance of Mice to Mycobacterium paratuberculosis Is Influenced by Slc11a1 at the Early but Not at the Late Stage of Infection▿  
Infection and Immunity  2008;76(5):2099-2105.
We have recently described the development of a luminescent Mycobacterium paratuberculosis strain of bovine origin expressing the luxAB genes of Vibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect of Slc11a1 (formerly Nramp1) polymorphism on susceptibility to M. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as for Mycobacterium avium subsp. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c, congenic BALB.B10-H2b (BALB/c background; H-2b), C57BL/6, and beige C57BL/6bg/bg mice (all Slc11a1s), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both Slc11a1r) and in (C57BL/6 × DBA/2)F1 mice (Slc11a1s/r), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bg mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.
PMCID: PMC2346681  PMID: 18285491
15.  Improved Protective Efficacy of a Species-Specific DNA Vaccine Encoding Mycolyl-Transferase Ag85A from Mycobacterium ulcerans by Homologous Protein Boosting 
Vaccination with plasmid DNA encoding Ag85A from M. bovis BCG can partially protect C57BL/6 mice against a subsequent footpad challenge with M. ulcerans. Unfortunately, this cross-reactive protection is insufficient to completely control the infection. Although genes encoding Ag85A from M. bovis BCG (identical to genes from M. tuberculosis) and from M. ulcerans are highly conserved, minor sequence differences exist, and use of the specific gene of M. ulcerans could possibly result in a more potent vaccine. Here we report on a comparison of immunogenicity and protective efficacy in C57BL/6 mice of Ag85A from M. tuberculosis and M. ulcerans, administered as a plasmid DNA vaccine, as a recombinant protein vaccine in adjuvant or as a combined DNA prime-protein boost vaccine. All three vaccination formulations induced cross-reactive humoral and cell-mediated immune responses, although species-specific Th1 type T cell epitopes could be identified in both the NH2-terminal region and the COOH-terminal region of the antigens. This partial species-specificity was reflected in a higher—albeit not sustained—protective efficacy of the M. ulcerans than of the M. tuberculosis vaccine, particularly when administered using the DNA prime-protein boost protocol.
Author Summary
Buruli ulcer (BU) is an infectious disease characterized by deep, ulcerating skin lesions, particularly on arms and legs, that are provoked by a toxin. BU is caused by a microbe belonging to the same family that also causes tuberculosis and leprosy. The disease is emerging as a serious health problem, especially in West Africa. Vaccines are considered to be the most cost-effective strategy to control and eventually eradicate an infectious disease. For the moment, however, there is no good vaccine against BU, and it is still not fully understood which immune defence mechanisms are needed to control the infection. The identification of microbial components that are involved in the immune control is an essential step in the development of an effective vaccine. In this paper, we describe the identification of one of these microbial components, i.e., antigen 85A, a protein involved in the integrity of the cell wall of the microbe. Our findings obtained in a mouse model now need to be extended to other experimental animals and later to humans. Combination with a vaccine targeting the toxin may be a way to strengthen the effectiveness of the vaccine.
PMCID: PMC2265439  PMID: 18350112
16.  Lack of Immune Responses to Mycobacterium tuberculosis DosR Regulon Proteins following Mycobacterium bovis BCG Vaccination▿  
Infection and Immunity  2007;75(7):3523-3530.
Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB), despite its variable protective efficacy. Relatively little is known about the immune response profiles following BCG vaccination in relation to protection against TB. Here we tested whether BCG vaccination results in immune responses to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens are targeted by the immune system during persistent Mycobacterium tuberculosis infection and have been associated with immunity against latent M. tuberculosis infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis showed at least 97% amino acid sequence homology, with 41 out of 48 genes being identical. Transcriptional profiling of 14 different BCG strains, under hypoxia and nitric oxide exposure in vitro, revealed a functional DosR regulon similar to that observed in M. tuberculosis. Next, we assessed human immune responses to a series of immunodominant TB latency antigens and found that BCG vaccination fails to induce significant responses to latency antigens. Similar results were obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency antigens were observed in individuals with suspected exposure to TB (as indicated by positive gamma interferon responses to TB-specific antigens ESAT-6 and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency antigens. Since immune responses to TB latency antigens have been associated with control of latent M. tuberculosis infection, our findings support the development of vaccination strategies incorporating DosR regulon antigens to complement and improve the current BCG vaccine.
PMCID: PMC1932964  PMID: 17502400
17.  A Booster Vaccination with Mycobacterium bovis BCG Does Not Increase the Protective Effect of the Vaccine against Experimental Mycobacterium ulcerans Infection in Mice▿  
Infection and Immunity  2007;75(5):2642-2644.
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.
PMCID: PMC1865750  PMID: 17325046
18.  Immunogenicity of Eight Dormancy Regulon-Encoded Proteins of Mycobacterium tuberculosis in DNA-Vaccinated and Tuberculosis-Infected Mice▿  
Infection and Immunity  2006;75(2):941-949.
Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F1 mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.
PMCID: PMC1828490  PMID: 17145953
19.  Development of Luminescent Mycobacterium avium subsp. paratuberculosis for Rapid Screening of Vaccine Candidates in Mice  
Infection and Immunity  2006;74(6):3684-3686.
Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.
PMCID: PMC1479266  PMID: 16714604
20.  Partial Reconstitution of the CD4+-T-Cell Compartment in CD4 Gene Knockout Mice Restores Responses to Tuberculosis DNA Vaccines  
Infection and Immunity  2006;74(5):2751-2759.
Reactivation tuberculosis (TB) is a serious problem in immunocompromised individuals, especially those with human immunodeficiency virus (HIV) coinfection. The adaptive immune response mediated by CD4+ and CD8+ T cells is known to confer protection against TB. Hence, vaccines against TB are designed to activate these two components of the immune system. Anti-TB DNA vaccines encoding the immunodominant proteins Ag85A, Ag85B, and PstS-3 from Mycobacterium tuberculosis are ineffective in mice lacking CD4+ T cells (CD4−/− mice). In this study, we demonstrate that reconstitution of the T-cell compartment in CD4−/− mice restores vaccine-specific antibody and gamma interferon (IFN-γ) responses to these DNA vaccines. The magnitude of the immune responses correlated with the extent of reconstitution of the CD4+-T-cell compartment. Reconstituted mice vaccinated with DNA encoding PstS-3, known to encode a dominant Db-restricted CD8+-T-cell epitope, displayed CD8+-T-cell responses not observed in CD4−/− mice. M. tuberculosis challenge in reconstituted mice led to the extravasation of IFN-γ-producing CD4+ and CD8+ T cells into lungs, the primary site of bacterial replication. Importantly, a reconstitution of 12 to 15% of the CD4+-T-cell compartment resulted in Ag85B plasmid DNA-mediated protection against a challenge M. tuberculosis infection. Our findings provide evidence that anti-TB DNA vaccines could be effective in immunodeficient individuals after CD4+-T-lymphocyte reconstitution, as may occur following antiretroviral therapy in HIV+ patients.
PMCID: PMC1459720  PMID: 16622212
21.  Immune Responses Induced in Cattle by Vaccination with a Recombinant Adenovirus Expressing Mycobacterial Antigen 85A and Mycobacterium bovis BCG  
Infection and Immunity  2006;74(2):1416-1418.
Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.
PMCID: PMC1360309  PMID: 16428796
22.  Members of the 30- to 32-Kilodalton Mycolyl Transferase Family (Ag85) from Culture Filtrate of Mycobacterium avium subsp. paratuberculosis Are Immunodominant Th1-Type Antigens Recognized Early upon Infection in Mice and Cattle  
Infection and Immunity  2006;74(1):202-212.
The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-γ) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-γ responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.
PMCID: PMC1346609  PMID: 16368974
24.  Buruli Ulcer (M. ulcerans Infection): New Insights, New Hope for Disease Control 
PLoS Medicine  2005;2(4):e108.
Buruli ulcer is a disease of skin and soft tissue caused by Mycobacterium ulcerans. It can leave affected people scarred and disabled. What are the prospects for disease control?
PMCID: PMC1087202  PMID: 15839744
25.  Mycobacterium tuberculosis with Disruption in Genes Encoding the Phosphate Binding Proteins PstS1 and PstS2 Is Deficient in Phosphate Uptake and Demonstrates Reduced In Vivo Virulence  
Infection and Immunity  2005;73(3):1898-1902.
By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.
PMCID: PMC1064925  PMID: 15731097

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