A Mycobacterium ulcerans vaccine pilot trial using an accurate low-dose challenge.

A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Multi-epitope vaccine candidates based on mycobacterial membrane protein large (MmpL) proteins against Mycobacterium ulcerans.

Buruli ulcer (BU) is a neglected tropical disease. It is caused by the bacterium Mycobacterium ulcerans and is characterized by skin lesions. Several studies were performed testing the Bacillus Calmette-Guérin (BCG) vaccine in human and animal models and M. ulcerans-specific vaccines in animal models. However, there are currently no clinically accepted vaccines to prevent M. ulcerans infection. The aim of this study was to identify T-cell and B-cell epitopes from the mycobacterial membrane protein large (MmpL) proteins of M. ulcerans. These epitopes were analysed for properties including antigenicity, immunogenicity, non-allergenicity, non-toxicity, population coverage and the potential to induce cytokines. The final 8 CD8+, 12 CD4+ T-cell and 5 B-cell epitopes were antigenic, non-allergenic and non-toxic. The estimated global population coverage of the CD8+ and CD4+ epitopes was 97.71%. These epitopes were used to construct five multi-epitope vaccine constructs with different adjuvants and linker combinations. The constructs underwent further structural analyses and refinement. The constructs were then docked with Toll-like receptors. Three of the successfully docked complexes were structurally analysed. Two of the docked complexes successfully underwent molecular dynamics simulations (MDS) and post-MDS analysis. The complexes generated were found to be stable. However, experimental validation of the complexes is required.

Comprehensive Case-Control Study of Protective and Risk Factors for Buruli Ulcer, Southeastern Australia.

To examine protective and risk factors for Buruli ulcer (BU), we conducted a case-control study of 245 adult BU cases and 481 postcode-matched controls across BU-endemic areas of Victoria, Australia. We calculated age- and sex-adjusted odds ratios for socio-environmental, host, and behavioral factors associated with BU by using conditional logistic regression. Odds of BU were >2-fold for persons with diabetes mellitus and persons working outdoors who had soil contact in BU-endemic areas (compared with indoor work) but were lower among persons who had bacillus Calmette-Guérin vaccinations. BU was associated with increasing numbers of possums and with ponds and bore water use at residences. Using insect repellent, covering arms and legs outdoors, and immediately washing wounds were protective; undertaking multiple protective behaviors was associated with the lowest odds of BU. Skin hygiene/protection behaviors and previous bacillus Calmette-Guérin vaccination might provide protection against BU in BU-endemic areas.

A human model of Buruli ulcer: The case for controlled human infection and considerations for selecting a Mycobacterium ulcerans challenge strain.

Critical knowledge gaps regarding infection with Mycobacterium ulcerans, the cause of Buruli ulcer (BU), have impeded development of new therapeutic approaches and vaccines for prevention of this neglected tropical disease. Here, we review the current understanding of host-pathogen interactions and correlates of immune protection to explore the case for establishing a controlled human infection model of M. ulcerans infection. We also summarise the overarching safety considerations and present a rationale for selecting a suitable challenge strain.

Identification of potential candidate vaccines against Mycobacterium ulcerans based on the major facilitator superfamily transporter protein.

Buruli ulcer is a neglected tropical disease that is characterized by non-fatal lesion development. The causative agent is Mycobacterium ulcerans (M. ulcerans). There are no known vectors or transmission methods, preventing the development of control methods. There are effective diagnostic techniques and treatment routines; however, several socioeconomic factors may limit patients' abilities to receive these treatments. The Bacillus Calmette-Guérin vaccine developed against tuberculosis has shown limited efficacy, and no conventionally designed vaccines have passed clinical trials. This study aimed to generate a multi-epitope vaccine against M. ulcerans from the major facilitator superfamily transporter protein using an immunoinformatics approach. Twelve M. ulcerans genome assemblies were analyzed, resulting in the identification of 11 CD8+ and 7 CD4+ T-cell epitopes and 2 B-cell epitopes. These conserved epitopes were computationally predicted to be antigenic, immunogenic, non-allergenic, and non-toxic. The CD4+ T-cell epitopes were capable of inducing interferon-gamma and interleukin-4. They successfully bound to their respective human leukocyte antigens alleles in in silico docking studies. The expected global population coverage of the T-cell epitopes and their restricted human leukocyte antigens alleles was 99.90%. The population coverage of endemic regions ranged from 99.99% (Papua New Guinea) to 21.81% (Liberia). Two vaccine constructs were generated using the Toll-like receptors 2 and 4 agonists, LprG and RpfE, respectively. Both constructs were antigenic, non-allergenic, non-toxic, thermostable, basic, and hydrophilic. The DNA sequences of the vaccine constructs underwent optimization and were successfully in-silico cloned with the pET-28a(+) plasmid. The vaccine constructs were successfully docked to their respective toll-like receptors. Molecular dynamics simulations were carried out to analyze the binding interactions within the complex. The generated binding energies indicate the stability of both complexes. The constructs generated in this study display severable favorable properties, with construct one displaying a greater range of favorable properties. However, further analysis and laboratory validation are required.

Vaccination efforts for Buruli ulcer.

BACKGROUND: Buruli ulcer is one of the most common mycobacterial diseases usually affecting poorer populations in tropical and subtropical environments. This disease, caused by M. ulcerans infection, has devastating effects for patients, with significant health and economic burden. Antibiotics are often used to treat affected individuals, but in most cases, surgery is necessary. AREA COVERED: We present progress on Buruli ulcer vaccines and identify knowledge gaps in this neglected tropical disease. EXPERT OPINION: The lack of appropriate infrastructure in endemic areas, as well as the severity of symptoms and lack of noninvasive treatment options, highlights the need for an effective vaccine to combat this disease. In terms of humoral immunity, it is vital to consider its significance and the magnitude to which it inhibits or slows down the progression of the disease. Only by answering these key questions will it be possible to tailor more appropriate vaccination and preventative provisions.

The search for a Buruli Ulcer vaccine and the effectiveness of the Bacillus Calmette-Guérin vaccine.

Buruli Ulcer is a neglected tropical disease that is caused by Mycobacterium ulcerans. It is not fatal; however, it manifests a range of devastating symptoms on the hosts' bodies. Various drugs and treatments are available for the disease; however, they are often costly and have adverse effects. There is still much uncertainty regarding the mode of transmission, vectors, and reservoir. At present, there are no official vector control methods, prevention methods, or a vaccine licensed to prevent infection. The Bacillus Calmette-Guérin vaccine developed against tuberculosis has some effectiveness against M. ulcerans. However, it is unable to induce long-lasting protection. Various types of vaccines have been developed based specifically against M. ulcerans; however, to date, none has entered clinical trials or has been released for public use. Additional awareness and funding are needed for research in this field and the development of more treatments, diagnostic tools, and vaccines.

Understanding the transmission of Mycobacterium ulcerans: A step towards controlling Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rare but chronic debilitating skin and soft tissue disease found predominantly in West Africa and Southeast Australia. While a moderate body of research has examined the distribution of M. ulcerans, the specific route(s) of transmission of this bacterium remain unknown, hindering control efforts. M. ulcerans is considered an environmental pathogen given it is associated with lentic ecosystems and human-to-human spread is negligible. However, the pathogen is also carried by various mammals and invertebrates, which may serve as key reservoirs and mechanical vectors, respectively. Here, we examine and review recent evidence from these endemic regions on potential transmission pathways, noting differences in findings between Africa and Australia, and summarising the risk and protective factors associated with Buruli ulcer transmission. We also discuss evidence suggesting that environmental disturbance and human population changes precede outbreaks. We note five key research priorities, including adoption of One Health frameworks, to resolve transmission pathways and inform control strategies to reduce the spread of Buruli ulcer.

Systematic review of M. Bovis BCG and other candidate vaccines for Buruli ulcer prophylaxis.

Buruli ulcer, caused by Mycobacterium ulcerans, is a neglected tropical disease endemic to over 30 countries, with increasing incidence in temperate, coastal Victoria, Australia. Strategies to control transmission are urgently required. This study systematically reviews the literature to identify and describe candidate prophylactic Buruli ulcer vaccines. This review highlights that Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine studied in randomised controlled trials and confirms its importance as a benchmark for comparison against putative vaccines in pre-clinical studies. Nevertheless, BCG alone is unable to offer long-term protection in humans. A number of experimental vaccines that exceed the protection provided by BCG in mice have emerged, particularly those utilising recombinant BCG expressing immunogenic M. ulcerans proteins. Although progress is promising, there remain key questions about the optimal approach to characterising the immunological correlates of protection in humans and strategies to investigate the safety and efficacy of such vaccines in humans.

Ketogenic Diet Impairment of Mycobacterium ulcerans Growth and Toxin Production and Enhancement of Host Response to Infection in an Experimental Mouse Model.

Ketogenic diets have been used to treat diverse conditions, and there is growing evidence of their benefits for tissue repair and in inflammatory disease treatment. However, their role in infectious diseases has been little studied. Buruli ulcer (Mycobacterium ulcerans infection) is a chronic infectious disease characterized by large skin ulcerations caused by mycolactone, the major virulence factor of the bacillus. In the current study, we investigated the impact of ketogenic diet on this cutaneous disease in an experimental mouse model. This diet prevented ulceration, by modulating bacterial growth and host inflammatory response. ß-hydroxybutyrate, the major ketone body produced during ketogenic diet and diffusing in tissues, impeded M. ulcerans growth and mycolactone production in vitro underlying its potential key role in infection. These results pave the way for the development of new patient management strategies involving shorter courses of treatment and improving wound healing, in line with the major objectives of the World Health Organization.

Bacille Calmette Guérin (BCG) and new TB vaccines: Specific, cross-mycobacterial and off-target effects.

The Bacille Calmette Guérin (BCG) vaccine was developed over a century ago and has become one of the most used vaccines without undergoing a modern vaccine development life cycle. Despite this, the vaccine has protected many millions from severe and disseminated forms of tuberculosis (TB). In addition, BCG has cross-mycobacterial effects against non-tuberculous mycobacteria and off-target (also called non-specific or heterologous) effects against other infections and diseases. More recently, BCG's effects on innate immunity suggest it might improve the immune response against viral respiratory infections including SARS-CoV-2. New TB vaccines, developed over the last 30 years, show promise, particularly in prevention of progression to disease from TB infection in young adults. The role of BCG in the context of new TB vaccines remains uncertain as most participants included in trials have been previously BCG immunised. BCG replacement vaccines are in efficacy trials and these may also have off-target effects.

Impact of common skin diseases on children in rural Côte d'Ivoire with leprosy and Buruli ulcer co-endemicity: A mixed methods study.

BACKGROUND: Skin-related neglected tropical diseases (skin NTDs) occur against a background of a very high prevalence of common skin diseases in sub-Saharan Africa. In this study, we examined the knowledge, attitude and practices (KAP) and the impact of common skin diseases in children living in a leprosy and Buruli ulcer (BU) co-endemic district in a west African country of Côte d'Ivoire, in order to help inform disease control efforts for skin NTDs. METHODS AND PRINCIPLE FINDINGS: Fourteen focus group discussions (FGDs) with schoolchildren, 5 FGDs with parents of a child affected with skin disease(s), and 27 in-depth semi-structured interviews with key personnel were conducted. The Children's Dermatology Quality of Life Index (CDLQI) questionnaire was applied to 184 schoolchildren with skin diseases. We found that there was ignorance or neglect towards skin diseases in general, due to their high prevalence and also the perceived minimal impact on children's daily lives. While the median score for the CDLQI questionnaire was 5 (IQR 2-9) out of 30, a range of scores was observed. Symptoms such as pruritus and experiencing bullying by classmates contributed to reduction in their quality of life. Poor hygiene was considered as a major cause of skin diseases. CONCLUSIONS/SIGNIFICANCE: Despite their high impact on affected populations, we observed a high level of ignorance and neglect toward common skin diseases. There is a critical need to increase awareness of skin diseases, or skin health promotion, which supports changing of the health-seeking behaviour for skin conditions. This will aid in early detection and treatment of the skin NTDs, in addition to providing benefits for those affected by other skin diseases. Educational opportunities should be utilized to their utmost. One would be associated with water, sanitation, and hygiene (WASH) strategies, but careful messages need to be developed and delivered.

Structural basis and designing of peptide vaccine using PE-PGRS family protein of Mycobacterium ulcerans-An integrated vaccinomics approach.

Buruli ulcer is an emerging tissue-necrosis infectious disease, caused by the pathogen Mycobacterium ulcerans, leading to permanent deformity if untreated. Despite this debilitating condition, no specific disease-modifying therapeutics or vaccination is available to date. Therefore, we aimed to design an effective multi-epitope vaccine against M. ulcerans using vaccinomics approach. Briefly, the highest antigenic PE-PGRS protein was selected from which the promiscuous T- and B-cell epitopes were predicted. After rigorous assessment, 15 promising T- and B-cell epitopes were selected. The identified T-cell epitopes showed marked interactions towards their HLA-binding alleles and provided 99.8 % world population coverage. Consequently, a vaccine chimera was designed by connecting these epitopes with suitable linkers and LprG adjuvant. The vaccine construct was highly antigenic, immunogenic and non-allergenic; hence, subjected to homology modelling. The molecular docking and dynamics simulation revealed a strong and stable interaction between vaccine and toll-like receptor 2. The binding energy and dissociation constant were -15.3 kcal/mol and 5.9 × 10-12 M, respectively. The computer-simulated immune responses showed abundance of immunoglobulins, increased interferon-γ production, and macrophages activation which are crucial for immune response against M. ulcerans. Furthermore, disulfide bridging and in silico cloning were also performed. These results suggest that the vaccine, if validated experimentally, will be a promising candidate against M. ulcerans and prevent Buruli ulcer disease.

Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

Effect of well drilling on Buruli ulcer incidence in Benin: a case-control, quantitative survey.

BACKGROUND: Buruli ulcer is the third most common mycobacterial disease worldwide. The public health burden of this neglected tropical disease is large, particularly in poor areas of west and central Africa. The development of appropriate preventive strategies is hampered by an incomplete understanding of the epidemiology and transmission of the disease. We investigated the effect of the drilling of wells on Buruli ulcer incidence. METHODS: In this case-control, quantitative survey, we obtained field data for Buruli ulcer incidence over a 10-year period from a specialised centre that collected data for the Ouémé and Plateau departments in Benin, and data for well drilling from the Ministry of Energy, Water and Mines in Benin. The coordinates of the wells drilled were obtained during site visits. A case-control study was then done to investigate the role of well water use in protecting against Buruli ulcer. FINDINGS: We found a strong inverse correlation between the incidence of Buruli ulcer and the number of new wells drilled in the Bonou municipality (r2=0·8818). A case-control study (106 cases and 212 controls) showed that regular use of the water from the wells for washing, bathing, drinking, or cooking was protective against Buruli ulcer (adjusted odds ratio 0·1, 95% CI 0·04-0·44; p=0·0012). INTERPRETATION: This study opens up new possibilities for developing an effective yet affordable policy to fight the disease on a substantial geographical scale. Our study shows that providing access to protected water is an efficient and feasable way to reduce the incidence of Buruli ulcer. FUNDING: Fondation Francaise Raoul Follereau, French National Institute of Health and Medical Research, and Région Pays de Loire.

Buruli ulcer in southern Côte D'ivoire: dynamic schemes of perception and interpretation of modes of transmission.

Buruli ulcer (BU) belongs to the group of neglected tropical diseases and constitutes a public health problem in many rural communities in Côte d'Ivoire. The transmission patterns of this skin infection are poorly defined, hence the current study aimed to contribute to the understanding, perceptions and interpretations of its mode of transmission using a socio-environmental approach. Social and environmental risk factors that may expose people to infection, and the dynamics of local transfer of knowledge and practices related to BU, were assessed in two endemic locations in southern Côte d'Ivoire, i.e. Taabo and Daloa. Data were generated by the administration of a household questionnaire (N=500) between February and June 2012 to assess how the population perceived transmission of BU, focus group discussions with local communities (N=8) to analyse ideologies regarding transmission patterns and semi-structured interviews with patients or their parents, former BU patients and traditional healers (N=30). The interviewees' empirical knowledge of the disease was found to be close to its biomedical description. Their aetiological perception of the disease was linked to natural (e.g. dirty water, insects) and supernatural (e.g. witchcraft, fate) causes. Some informants attributed the spread of the disease to recently immigrated neighbouring communities whose arrival coincided with an increase in reported BU cases. However, the general consensus seemed to be that the main mode of transmission was contact with infested soil or ulcerated wounds. The participants were aware that BU was a socio-environmental problem in these endemic areas, offering a good starting point for educational campaigns for at-risk communities. Buruli ulcer control programmes should therefore include educational campaigns and Water, Sanitation and Hygiene (WASH) interventions for those at risk in affected communities.

Integrated approach in the control and management of skin neglected tropical diseases in Lalo, Benin.

BACKGROUND: Neglected Tropical Diseases (NTDs) are a group of several communicable diseases prevalent in the tropical and subtropical areas. The co-endemicity of these diseases, the similarity of the clinical signs, and need to maximize limited financial and human resources have necessitated implementation of integrated approach. Our study aims to share the lessons of this integrated approach in the fight against Buruli ulcer (BU), leprosy and yaws in a rural district in Benin. METHODS: It is a cross-sectional study using a single set of activities data conducted from May 2016 to December 2016. Health workers and community health volunteers involved in this study were trained on integrated approach of the Buruli ulcer, leprosy and yaws. Village chiefs were briefed about the activity. The trained team visited the villages and schools in the district of Lalo in Benin. After the education and awareness raising sessions, all persons with a skin lesion who presented voluntarily to the team were carefully examined in a well-lit area which respected their privacy. Suspected cases were tested as needed. The socio-demographic information and the characteristics of the lesions were collected using a form. A descriptive analysis of the epidemiological, clinical and laboratory variables of the cases was made using Excel 2013 and SPSS version 22.00. PRINCIPAL FINDINGS: In the study period, 1106 people were examined. The median (IQR) age of those examined was 11 (8; 27) years. Of 34 (3.1%) suspected BU cases, 15 (1.4%) were confirmed by PCR. Only three cases of leprosy were confirmed. The 185 (16.7%) suspected cases of yaws were all negative with the rapid test. The majority of cases were other skin conditions, including fungal infections, eczema and traumatic lesions. CONCLUSION: The integrated approach of skin NTD allows optimal use of resources and surveillance of these diseases. Sustaining this skin NTD integrated control will require the training of peripheral health workers not only on skin NTD but also on basic dermatology.