Prospects for the development of a subunit vaccine against Mycobacterium ulcerans disease (Buruli ulcer)

Buruli ulcer (BU) is a slow progressing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. It presents in different clinical forms ranging from small non-ulcerative nodules to large ulcers and sometimes multiple ulcerations. The highest prevalence of the focally occurring disease is found in rural areas of West African countries. Both the mode of transmission as well as the potential environmental reservoir of M. ulcerans remain unidentified to date. In Cameroon, the remote Mapé dam region has recently been identified as a new BU endemic area. To assess the age-adjusted prevalence and local geographic distribution of BU, a house-by-house survey in the Bankim health district was conducted. Results showed that children between the age of five to 15 and elderly people were over proportionally affected by BU. To confirm the clinical diagnosis of BU during and after the health survey in Bankim, fine needle aspirates and swabs from undermined ulcer edges were transported to the Swiss Tropical and Public Health Institute for laboratory confirmation by quantitative real time PCR. In parallel we developed a protocol for primary culture initiation of M. ulcerans from patient lesions after a long time span between sampling and processing in a BSL3 culture laboratory. The established two sets of Cameroonian patient isolates from the Mapé and the Nyong river valleys were used for a comparative genome sequencing study revealing the presence of two distinct phylogenetic clonal complexes in Cameroon. Despite the fact that BU can be treated with antibiotics, the socioeconomic impact of the disease on affected populations remains devastating. As long as it is not clearly known how the disease is contracted, interruption of transmission is not an option. A vaccine against M. ulcerans on the other hand could be used both as preventive measure and therapeutically. While sero-epidemiological studies imply the presence of protective immunity in some individuals, no vaccine is available to date. Within the framework of the EU funded collaborative project BuruliVac we investigated the potential for developing a protein subunit vaccine against M. ulcerans by delivering vaccine candidate antigens with three different systems: i. as recombinant proteins with an adjuvant, ii. as vesicular stomatitis virus replicon and iii. incorporated into a genetically modified mouse malaria parasite (Plasmodium berghei) in an infection - treatment - approach. All three formulations were assessed for their immunogenicity and their protective potential in a mouse model of BU. Although all three vaccination approaches elicited strong humoral immune responses, no full protection was observed for any of the formulations. However a slight partial protection was seen for a replicon - prime / recombinant protein boost regimen with a vesicular stomatitis virus replicon incorporating an expression cassette for the M. ulcerans protein MUL2232. Additionally, a transient delay of foot pad swelling was observed in mice receiving infection - treatment - vaccination with P. berghei expressing MUL4987. Despite the mainly extracellular nature of M. ulcerans in infected tissue, antibody production against the protein vaccine candidates thus does not seem to be sufficient for protection. Considering marked differences between the mouse footpad model of BU and the disease in humans, we aimed at developing an animal model that better reflects local pathogenesis and host-pathogen interactions in the human BU lesions. Hence, we developed the pig as novel animal model for BU and showed that the observed histopathological changes in the infected pig skin closely represent those of human BU. Therefore the pig model has great potential for the validation of new therapeutic and prophylactic interventions

Dynamics of Mycobacterium tuberculosis Ag85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay.

Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. We generated new monoclonal antibodies that recognize the Mycobacterium tuberculosis secreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generated in vitro and in vivo We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in the M. tuberculosis complex: compared with the commonly used H37Rv strain (lineage 4), Mycobacterium africanum (lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCE Bacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions

Use of recombinant virus replicon particles for vaccination against Mycobacterium ulcerans disease

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans

Experimental infection of the pig with Mycobacterium ulcerans : a novel model for studying the pathogenesis of Buruli ulcer disease

Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available.; For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2×107 and 2×106 colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans.; Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions

Geographic distribution, age pattern and sites of lesions in a cohort of buruli ulcer patients from the mapé basin of cameroon

Buruli ulcer (BU), a neglected tropical disease of the skin, caused by Mycobacterium ulcerans, occurs most frequently in children in West Africa. Risk factors for BU include proximity to slow flowing water, poor wound care and not wearing protective clothing. Man-made alterations of the environment have been suggested to lead to increased BU incidence. M. ulcerans DNA has been detected in the environment, water bugs and recently also in mosquitoes. Despite these findings, the mode of transmission of BU remains poorly understood and both transmission by insects or direct inoculation from contaminated environment have been suggested. Here, we investigated the BU epidemiology in the Mapé basin of Cameroon where the damming of the Mapé River since 1988 is believed to have increased the incidence of BU. Through a house-by-house survey in spring 2010, which also examined the local population for leprosy and yaws, and continued surveillance thereafter, we identified, till June 2012, altogether 88 RT-PCR positive cases of BU. We found that the age adjusted cumulative incidence of BU was highest in young teenagers and in individuals above the age of 50 and that very young children (>5) were underrepresented among cases. BU lesions clustered around the ankles and at the back of the elbows. This pattern neither matches any of the published mosquito biting site patterns, nor the published distribution of small skin injuries in children, where lesions on the knees are much more frequent. The option of multiple modes of transmission should thus be considered. Analyzing the geographic distribution of cases in the Mapé Dam area revealed a closer association with the Mbam River than with the artificial lake

Experimental infection of the pig with Mycobacterium ulcerans: a novel model for studying the pathogenesis of Buruli ulcer disease.

BACKGROUND Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available. METHODOLOGY/PRINCIPAL FINDINGS For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2 × 10(7) and 2 × 10(6) colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans. CONCLUSION/SIGNIFICANCE Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer

YesBackground. Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology. We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings. The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions. To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.This work was supported by the UBS Optimus Foundation (Zurich, Switzerland) and the Department of Economy, Science and Innovation of the Flemish Government (Belgium). KV was supported by a VLADOC PhD scholarship of VLIRUOS (Belgium)

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Interferon-γ is a crucial activator of early host immune defense against Mycobacterium ulcerans infection in mice

Buruli ulcer (BU), caused by infection with Mycobacterium ulcerans, is a chronic necrotizing human skin disease associated with the production of the cytotoxic macrolide exotoxin mycolactone. Despite extensive research, the type of immune responses elicited against this pathogen and the effector functions conferring protection against BU are not yet fully understood. While histopathological analyses of advanced BU lesions have demonstrated a mainly extracellular localization of the toxin producing acid fast bacilli, there is growing evidence for an early intra-macrophage growth phase of M. ulcerans. This has led us to investigate whether interferon-γ might play an important role in containing M. ulcerans infections. In an experimental Buruli ulcer mouse model we found that interferon-γ is indeed a critical regulator of early host immune defense against M. ulcerans infections. Interferon-γ knockout mice displayed a faster progression of the infection compared to wild-type mice. This accelerated progression was reflected in faster and more extensive tissue necrosis and oedema formation, as well as in a significantly higher bacterial burden after five weeks of infection, indicating that mice lacking interferon-γ have a reduced capacity to kill intracellular bacilli during the early intra-macrophage growth phase of M. ulcerans. This data demonstrates a prominent role of interferon-γ in early defense against M. ulcerans infection and supports the view that concepts for vaccine development against tuberculosis may also be valid for BU