1,721,147 research outputs found
Overview: Mycobacterium ulcerans disease (buruli ulcer)
No full textEnhanced international research efforts since the establishment of the Global BU Initiative in 1998 by the WHO have helped to advance our understanding of the epidemiology, and pathogenesis of Mycobacterium ulcerans infections. Improved methods to cultivate the extremely slow-growing pathogen from BU lesions have laid the groundwork for a variety of studies using M. ulcerans isolates, including the analysis of the genome and proteome of the pathogen, as well as drug susceptibility testing and analyses of host-pathogen interactions in vitro and in animal models. The identification of specific, high-copy number target sequences in the genome of M. ulcerans has enabled the development of diagnostic tests and assays to detect the pathogen in the environment. Important research questions remain about the reservoir(s) of M. ulcerans in aquatic environments, factors leading to or promoting transmission to hosts, and host-pathogen interactions resulting in chronic infection versus spontaneous healing
Identification and characterization of "Plasmodium falciparum" and "Mycobacterium ulcerans" antigens as potential vaccine components and targets for serological test and molecular typing methods
Full text linkThe increase of Plasmodium falciparum’s drug resistance and the resurgence of
Mycobacterium ulcerans disease through environmental changes aggravate health
problems caused by these pathogens.
Buruli ulcer, caused by M. ulcerans, is after tuberculosis and leprosy the third most
common mycobacterial human infection and the most poorly understood of these three
diseases. It is characterized by chronic, necrotizing ulceration of subcutaneous tissues and
the overlying skin. M. ulcerans is a slow-growing mycobacterium which multiplies extracellularly
in Buruli ulcer lesions. There is a broad antigenic overlap between mycobacterial
species, which complicates the analysis of adaptive immune responses and hampers the
development of specific sero-diagnostic tests for M. ulcerans in areas where BCG
vaccination has been implemented and tuberculosis is endemic. In an effort to identify
immunodominant antigens of M. ulcerans, we have generated panels of monoclonal
antibodies from mice immunized with this pathogen. Cross-reactivity studies with other
mycobacterial species performed by Western blot and immunofluorescence assays have
identified immunodominant epitopes with a limited cross-species distribution (18kDa and
the 34-37kDa proteins). In contrast, the majority of antigens were spread widely amongst
different mycobacterial species. One set of non-crossreactive monoclonal antibodies
recognized an 18kDa protein of M. ulcerans that is associated with the cell-wall fraction,
and expressed in Buruli ulcer lesions. The target protein was identified by massspectroscopy
as the M. ulcerans orthologue of the M. leprae 18kDa small heat shock
protein, which has no orthologues in the genomes of M. bovis and M. tuberculosis. Human
anti-18kDa small heat shock protein antibodies were found in the serum of all Buruli ulcer
patients tested, but not in sera from Europeans volunteers and only rarely in sera from
Africans living in Buruli ulcer non-endemic regions. Reactivity of sera from a large
proportion of people living in a Buruli ulcer endemic area and in contact with Buruli ulcer
patients indicated that an 18kDa small heat shock protein-based serological test is suitable
to detect exposure to M. ulcerans.
Since M. ulcerans shows only very limited genetic diversity, standard multi-locus
sequence typing of housekeeping genes is not a suitable tool for molecular epidemiological
analysis of Buruli ulcer. Among the monoclonal antibodies exhibiting broad inter-species
cross-reactivity, one group recognized the M. ulcerans orthologue of mycobacterial
laminin-binding protein. DNA sequence analysis demonstrated that the corresponding
hupB gene from M. ulcerans isolates of diverse geographical origin exhibited considerable
diversity based both on insertional/deletional polymorphism and on single base exchanges.
Dominance of non-conservative exchanges was indicative of a diversifying selection
pressure. Sequences analysis of a set of such variable genes may develop into a new tool
for genetic fingerprinting of isolates.
There is great need to identify new malaria vaccine and drug targets. Monoclonal
antibodies were used to characterize a novel conserved protein of P. falciparum designated
D13. Western blot analysis demonstrated that D13 is stage-specifically expressed during
schizogony in asexual blood stages of the parasite. It has a functionally essential role in
parasite biology, since anti-D13 monoclonal antibodies have parasite growth inhibitory
activity. The D13 protein may represent a suitable target for a malaria vaccine design.
Immunofluorescence analysis with monoclonal antibodies specific for glyceraldehydes-3-
phosphate dehydrogenase (pfGAPDH) and pfAldolase showed that pfGAPDH and
pfAldolase colocalise in early stages of both liver and asexual blood stage parasite
development. However, during schizogony, unlike pfAldolase, pfGAPDH was enriched in
the apical region of the parasites. In addition, Western blot analyses demonstrate that
pfGAPDH is in both the membrane-containing pellet and supernatant fractions. These
results have provided evidence that pfGAPDH exerts non-glycolytic function(s) in P.
falciparum; including possibly a role in vesicular transport and biogenesis of apical
organelles. This data together with the limited amino acid sequence identity with human
GAPDH suggest that the pfGAPDH could be a promising safe target for drug treatment
Characterization of novel surface proteins of "Plasmodium falciparum" and their assessment as molecular targets for a malaria subunit vaccine
Full text linkEach year there are more than 250 million cases of malaria, claiming nearly one million deaths of which most are among children below the age of five from sub-Saharan Africa. An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. To date, no approved malaria vaccine is available and only a few candidate vaccines were able to induce some protective efficacy. Limited success in the development of a malaria vaccine may partly be due to the reliance on a hand full of antigens discovered more than 20 years ago. Since the fully annotated Plasmodium falciparum genome has become available in 2002, reverse vaccinology presents a new opportunity to identify novel malaria vaccine candidate antigens.
In this thesis, we anticipated the rational selection of novel malaria subunit vaccine candidates. Proteins on the surface of extracellular stages of the malaria parasite are accessible to immune surveillance by antibodies. Thus, our strategy is based on the selection of hypothetical surface proteins of extracellular parasite stages and subsequent functional characterization using specific monoclonal antibodies.
Functional in vitro and in vivo assays require antibodies capable of recognizing the endogenous antigen in its native context. We developed an entirely cell-based approach that bypasses the problematic step of protein purification. By presenting the antigen on the surface of mammalian cells in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of monoclonal antibodies capable of binding to the native endogenous target proteins. This was exemplified by three hypothetical surface proteins of P. falciparum, whereof two contain complex folds comprising numerous disulfide bonds. The developed new strategy for the generation of monoclonal antibodies may be applied for a wide range of cell-surface proteins.
The generated monoclonal antibodies were used to study the selected hypothetical P. falciparum surface proteins. PF14_0325 was found to be expressed in late asexual blood stages and PFF0620c, a member of the 6-cysteine protein family implicated in cell-cell interactions, in gametocytes and sporozoites. Both proteins await further investigation in assay systems assessing sporozoite and sexual blood stage inhibition. A third candidate, designated cysteine-rich protective antigen (CyRPA), is expressed in merozoites and localizes to its apical pole. CyRPA-specific antibodies inhibited parasite growth in vitro as well as in vivo. The inhibitory mechanism was found to be independent of immune cells or complement, although complement augmented the effect in vivo. Antibodies blocked invasion of merozoites into erythrocytes, indicating that CyRPA has a function in merozoite invasion. The protein was found to be highly conserved. Together, this renders CyRPA a candidate component for a malaria blood stage vaccine.
The in vivo growth inhibitory effect of antibodies specific for malaria P. falciparum blood stage antigens was assessed by passive immunisation experiments in P. falciparum infected NOD/scid-IL2Rγnull mice engrafted with human erythrocytes. In contrast to previously described passive immunisation studies in other SCID mouse lines, this model evinced a dose-response relationship. Thus we propose this model for comparison of the relative in vivo inhibitory potency of malaria specific antibodies.
Applying the principle of reverse vaccinology, we identified and characterized surface proteins of extracellular malaria stages. Thereby we identified CyRPA as target of merozoite invasion-inhibitory antibodies. We expect that characterization of further hypothetical parasite proteins with this strategy will identify additional vaccine candidate antigens from the extracellular stages of P. falciparum. This will increase the choice of vaccine antigens for incorporation into an effective multivalent, multi-stage malaria subunit vaccine
Aspects of the pathogenesis, immunity and treatment of Buruli ulcer
Full text linkBuruli ulcer (BU) is a neglected tropical disease of the skin and subcutaneous tissue caused by infection with Mycobacterium ulcerans. The disease has been reported from over 30 countries worldwide with the highest prevalence in rural areas of West African countries. Clinically, M. ulcerans disease presents in different forms, ranging from small non-ulcerative nodules to large ulcers. The pathology of BU is largely attributable to the production of mycolactone, a lipid-like macrolide exotoxin with cytotoxic and immunosuppressive characteristics. Although mycolactone-induced cytotoxicity and tissue necrosis are the key elements of BU pathogenesis, the molecular mechanisms underlying these processes remained to be elucidated.
Within the framework of this PhD thesis, we could show that mycolactone binds to the 12-kDa FK506-binding protein and acts as a potent inhibitor of both mTORC1 and mTORC2. Inhibition of mTORC2 results in inactivation of Akt and dephosphorylation and activation of the Akt-targeted transcription factor FoxO3. Subsequent up-regulation of the FoxO3 target gene Bim, a pro-apoptotic member of the Bcl-2 protein family, was observed both in vitro and in human BU lesions. Moreover, Bim knockout mice were able to contain M. ulcerans infection and did not develop necrotic lesions with large clusters of extracellular bacilli typical for BU, highlighting the pivotal role of Bim-promoted apoptosis for BU pathogenesis.
In addition, we aimed at characterizing the nature of immune defense mechanisms conferring protection against BU. Specifically, we focused on the role of interferon-γ and of cellular immune effector mechanisms during the early intracellular stage of M. ulcerans infection. We could show that interferon-γ is critical for early host immunity against M. ulcerans, since mice lacking this cytokine showed a faster increase in bacterial burden and an accelerated pathogenesis, indicative of a reduced capacity to kill the bacilli during the early intracellular stage of the infection.
Finally, we evaluated the activity of the new tuberculosis drug candidate Q203 against M. ulcerans using the BU mouse foot pad infection model. In this project, we were able to demonstrate that Q203 has a higher activity against M. ulcerans than rifampicin, and might therefore be suited to replace rifampicin as the first line therapeutic option in the future
Genetic diversity and immune evasion of bacterial pathogens
Full text linkWithin the framework of this thesis the genetic diversity of three bacterial pathogens, Mycobacterium ulcerans, Neisseria meningitidis and Streptococcus agalactiae was investigated. The aim of these analyses was to contribute to the understanding of how genetic properties of the pathogens contribute to immune evasion. Implications of the findings for vaccine design are discussed.
Mycobacterium ulcerans
Buruli ulcer is a disease of skin and soft tissue caused by the bacterial pathogen Mycobacterium ulcerans. M. ulcerans has recently diverged from an M. marinum progenitor through the acquisition of a virulence plasmid, lateral gene transfer and reductive evolution. Isolates of M. ulcerans deriving from different regions of the globe can be associated with two distinct lineages, either the ancestral or the classical lineage. Here, we show that the two copies of the esxB-esxA gene cluster present in the genome of M. marinum are both deleted from the genome of M. ulcerans strains belonging to the highly virulent classical lineage. Members of the ancestral M. ulcerans lineage instead retained copies of the esxB-esxA gene cluster. Additionally, the hspX gene was present in the strains of the ancestral lineage and absent in the classical lineage. Our results indicate that M. ulcerans is adapting to an environment that is screened by immune recognition mechanisms by loss of highly immunogenic proteins.
Neisseria meningitidis
Certain hypervirulent lineages of Neisseria meningitidis, a commensal of the human nasopharynx, are a major cause of meningitis and septicaemia. Here we have investigated subcapsular antigens of serogroup A Neisseria meningitidis strains isolated in the course of longitudinal colonization and disease surveys in the African meningitis belt. In the course of clonal waves of colonization and disease we observed no sequence diversification of the outer membrane proteins PorA, PorB
and FetA. In contrast, high variability in the expression of Opa proteins was observed due to changing numbers of pentamer repeats within the open reading frames of the four opa genes opaA, opaB, opaD and opaJ. Furthermore, we found some exchange of alleles of the opa genes OpaA and OpaJ by horizontal gene transfer. Herd immunity may thus be a stronger driving force for diversification of Opa proteins than for other outer membrane proteins.
Streptococcus agalactiae
While Streptococcus agalactiae, the group B streptococcus (GBS), is traditionally considered a neonatal pathogen, it is also emerging as a significant cause of morbidity in adults. Here we have analysed the population structure of GBS isolates, collected from carriers and clinical cases in Kenya. Multi-locus sequence typing differentiated the 173 strains analyzed into 22 sequence types (STs), including 5 novel STs. A close correlation between STs and distinct capsular serotypes was found with the disease isolates being more diversified with respect to both STs and capsular serotypes than carrier isolates. The STs and capsular serotypes most prevalent in Kenya were also commonly found in many other regions of the world.
In this investigation, the highest genetic variablility was found in our GBS collection. In the N. meningitidis isolates collected during clonal waves of meningococcal colonization and disease, there was a striking lack of diversification, with the exception of the opa genes. The most conserved bacterial pathogen in this study was M. ulcerans, where no genetic variability could be found within a geographic region.
No vaccines exist to date against M. ulcerans or S. agalactiae, and an affordable universal vaccine against N. meningitidis is urgently needed. The technological advances in whole genome sequencing are likely to facilitate efforts towards finding suitable candidate antigens for subunit vaccines
Monoclonal antibodies as tools in antigen detection assay and vaccine development : design of a sensitive detection test for Brucella bacteria and profiling of the malaria vaccine candidate antigen reticulocyte-binding homolog 2 (PfRH2)
Full text linkWe aimed at identifying immunodominant Brucella antigens for implementation in new detection tools or for subunit vaccine development. In particular, our strategy was to produce Brucella cell surface antigen-specific monoclonal antibodies (mAbs) for the development of an antigen capture assay for the detection of Brucella cells as potential bio threat agents in complex samples. We generated a panel of Brucella lipopolysaccharide (LPS)-specific mAbs by immunising mice with inactivated B. melitensis and B. abortus cells. The mAbs recognised Brucella species with ‘smooth’ LPS independently of the way how the bacterial cells were inactivated. Two mAbs were implemented into a bead-based Luminex assay detecting ‘smooth’ Brucella spp. with species-dependent detection limits of 2 x 102 to 8 x 104 cells per mL. Integration of the Luminex assay into a multiplex format enabled simultaneous detection of Brucella spp. and three other bio threat agents within a single sample. The developed Luminex assay may be applied for the detection of whole Brucella cells both in natural Brucella outbreak and in bioterrorism attack scenarios.
We also tried to generate mAbs against Brucella cell surface proteins from mice immunised with inactivated whole Brucella cells. While serum antibody responses against both LPS and protein antigens were seen in Western blotting analyses, attempts to generate protein-specific mAbs failed, most likely due to the immunodominant nature of the LPS. Western blot analyses with Brucella lysate also identified antibodies against some immunodominant Brucella proteins in the serum of cattle naturally infected with Brucella spp., however, identification of the recognised proteins with a Brucella-specific peptide microarray failed.
In a second part of the thesis we aimed at evaluating the potential of the Plasmodium falciparum reticulocyte-binding homolog 2 (PfRH2) present in the rhoptries as a malaria blood stage vaccine antigen. We produced PfRH2-specific mAbs by immunising mice with the 40kDa receptor-binding domain of PfRH2. The PfRH2-specific mAbs cross-reacted with the natural PfRH2 protein present in schizont stage parasites and showed a rhoptry-characteristic staining pattern in immunofluorescence microscopy. However when evaluated in functional in vitro and in vivo assays PfRH2 specific mAbs showed no inhibitory effect on erythrocyte invasion. Furthermore, the invasion-inhibitory effect of mAbs specific for the cysteine-rich protective antigen (PfCyRPA) was not enhanced by PfRH2-specific mAbs
Skin neglected tropical diseases in Cameroon : the need for integrated control and elimination
Full text linkAmong the updated WHO list of 20 Neglected Tropical Diseases, about six manifests on the skin and are now known as Skin-NTDs. NTDs have not received equal attention from the international community compared to HIV/AIDS, malaria and tuberculosis although their burdens in terms of disability adjusted life years (DALYs) are comparable. Following advocacy for over a decade and half, NTDs are now being prioritized on the global health agenda, and the WHO has set a 2020 roadmap for accelerating work to overcome the global impact of NTDs.
For this PhD thesis, we aimed at determining the current burden of major skin-NTDs in Cameroon and make recommendations for proper control strategies. The basis was surveillance data from 2000 to 2014 for leprosy and Buruli ulcer available at the National Control Programme office, community-based surveys and a literature review.
We confirmed that Cameroon attained elimination of leprosy as a public health problem in 2000. Between 2000 and 2014, the leprosy prevalence and the detection dropped significantly, with the steepest reductions occurring between 2000 and 2005, followed by a stagnation from 2006 to 2014. We also showed a persistent but moderate transmission of leprosy, with an increasing trend between 2007 and 2014. Ten health districts had not achieved leprosy elimination, and eighteen were high leprosy-burdened according to the Leprosy Burden Score at the end of 2014. The increasing trend in leprosy transmission and the persistence of high-burdened districts were attributed to reduction in key leprosy control activities secondary to waning of resource allocation by the government and support partners. Some 3700 Buruli ulcer (BU) cases, with an annual average of 264 cases were treated from 2001 to 2014 in Cameroon. Control activities began in two endemic foci of Ayos and Akonolinga in the centre region and were later expanded to Ngoantet-Mbalmayo in the centre, Bankim in the Adamawa and Mbonge in the southwest regions following a national survey in 2004. Analysis of data from treatment centres created at these foci, further revealed presence of BU in 64 health districts mainly from the southern part of the country. BU case-detection increased between 2001 and 2005, and then declined progressively until 2014 and beyond. Analysis of key BU control indicators showed deterioration from 2010 to 2014 and beyond. We also highlighted the importance of differential diagnosis in a context of co-endemicity of mycobacterial diseases, through a case-report of cutaneous tuberculosis misdiagnosed for BU. BU activities in Cameroon were supported by two partners, who from 2010, reduce their support significantly to complete withdrawal of one of them in 2014. Currently there is little funding from the government budget, which is insignificant compared to the expressed needs. We also confirmed the resurgence of yaws in Cameroon through a survey among the pygmy population in the east region, and building on this, we have determined the status of yaws in 53 of 189 health districts and confirmed 37 of them as endemic for yaws.
Research conducted within the framework of this thesis, has increased our understanding of skin-NTDs, the efforts made in their control, and the challenges faced by the control activities in Cameroon. Based on these, we have recommended an integrated surveillance and control strategy of skin-NTDs to the National Control Programme. To the scientific community we recommend, accelerated research and development activities for point-of-care diagnostic tests, shorter treatment courses, and vaccines for reinforcement of prevention of these NTDs
Buruli Ulcer
Full text linkA major objective of this open access book is to summarize the current status of Buruli Ulcer (BU) research for the first time. It will identify gaps in our knowledge, stimulate research and support control of the disease by providing insight into approaches for surveillance, diagnosis, and treatment of Buruli Ulcer. Book chapters will cover the history, epidemiology diagnosis, treatment and disease burden of BU and provide insight into the microbiology, genomics, transmission and virulence of Mycobacterium ulcerans. ; Supports further investigation by summarizing state of the art in the field of Buruli ulcer research Enriches understanding of epidemiology of Buruli ulcer in different geographic regions Reviews exhaustively the characteristics of Mycobacterium ulcerans diseas
Epidemiology of Mycobacterium ulcerans disease in the Bankim Health Distrit of Cameroon and monitoring of the healing process of Buruli Ulcer lesions
Full text linkBuruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans which, if untreated, can lead to extensive tissue destruction and ulceration. The disease has been reported from over 30 countries with the highest prevalence in West Africa. Generally it is assumed that M. ulcerans is acquired from environmental sources, but BU is considered a “mysterious disease” because the natural reservoir and the mode of transmission are still not identified. Clinically BU presents with a spectrum of forms ranging from non-ulcerative lesions to large ulcers. The gold standard for diagnosing BU is IS2404 qPCR, which is a sophisticated technology not applicable in the field, where BU is often diagnosed on the basis of clinical signs and symptoms only. Direct microscopic smear examination after Ziehl-Neelsen staining, which has a low sensitivity, is the only point-of-care laboratory diagnostic method currently available. Since 2004, the WHO recommends to treat BU with a combination of streptomycin and rifampicin daily for 8 weeks. While this specific treatment is highly effective in killing the bacteria, healing of large ulcers may require long periods of time.
The Bankim Health District (HD) in the Mapé dam basin of Cameroon has been recently identified as BU endemic area and a new BU field research site was established by us in 2010. Within the framework of this thesis, we have contributed to strengthening of the local BU treatment and research site by the implementation of a surveillance and documentation system to promote a continuous case detection and follow up of patients, to investigate the pathway of transmission and to perform a comprehensive spatio-temporal distribution analysis of BU in the area.
Local clinical and microscopic diagnosis was re-confirmed by qPCR, bacterial culture and histopathology performed in Basel. The in-depth analysis on 148 qPCR confirmed cases underlined that BU is a pediatric disease in Africa and that the lesions occur mainly at the limbs with no differences amongst males and females. We obtained information on the exact geographical origin of 136 qPCR positive BU patients through mapping of their houses and farms. Results revealed for the majority of patients residence or agricultural activities close to the Mbam river. Sites of environmental contact of BU patients were screened to search for potential reservoirs of M. ulcerans. At one village water site, DNA of M. ulcerans, was persistently found over more than 2 years, indicating that the pathogen may persist in detritus.
Because some of the BU lesions healed very fast, while others showed an impaired healing process, we analyzed tissue samples in detail for the presence of wound healing and scarring biomarkers. Using the histopathological approach, we evaluated the use of markers of cell activation, myofibroblast formation and matrix deposition for the monitoring of the healing of BU lesions. While α-smooth muscle actin-positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as procollagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype. Healing wounds showed dermal tissue remodelling by apoptosis, and increased cytokeratin 16 expression in the epidermis.
Taken together, the results described in this thesis were obtained by a multidisciplinary approach. They contribute to our understanding of BU epidemiology and transmission, as well as of pathogenesis, wound healing and may eventually help to improve diagnosis, treatment and prevention of BU
- …
