Abstract
Buruli Ulcer (BU) is a neglected, necrotizing skin disease, caused by M. ulcerans, that can leave patients with prominent scars and lifelong disability. M. ulcerans produces a diffusible lipid toxin, mycolactone, essential for bacterial virulence. Prevention is difficult as little is known about disease transmission and there is no vaccine. There have been several recent advances in the field. These include sequencing of the bacterial genome and of the giant plasmid responsible for mycolactone synthesis, better understanding of the bacterial lifecycle and of the mechanism of action of the toxin. This work has revealed a number of possible vaccine candidates, some of which are shared with other mycobacteria, e.g. M. tuberculosis, while other targets are unique to M. ulcerans. In this review, we discuss several M. ulcerans vaccine targets and vaccination methods, and outline some of the gaps in our understanding of the bacterium and the immune response against it.
www.wiv-isp.be/Programs/communicable-infectious-diseases/Pages/EN-Immunology.aspx
Introduction
Buruli Ulcer (BU) is a necrotizing bacterial skin disease caused by Mycobacterium ulcerans. M. ulcerans produces a diffusible macrolide toxin, called mycolactone (ML), which is essential for bacterial virulence. BU has been documented in over 30 countries worldwide, although most of the cases occur in West Africa, primarily Benin, Côte d’Ivoire and Ghana.Citation1 According to the World Health Organization (http://apps.who.int/neglected_diseases/ntddata/buruli/buruli.html), there were 4,888 new BU cases reported in 2010, primarily in children under 15 y. However, as the disease is not notifiable in many countries and most patients live in remote, rural areas with little medical infrastructure, the actual number of cases is likely to be much higher. Regardless, as the disease burden is mostly localized to certain geographical areas, the impact of vaccination and treatment efforts can be very high.
Despite this, the disease has received limited attention by the medical and scientific communities as it is rarely lethal. However, morbidity associated with BU is high as extensive skin damage can leave patients with prominent scars, disfiguration and even amputated limbs. This has prompted a global effort, including the Global Buruli Ulcer Initiative (www.who.int/buruli/en/) and BuruliVac (www.burulivac.eu/), to improve detection, treatment and prevention of BU.
Prevention of BU is complicated by the fact that while most infected people live near lakes, rivers and swamps, where the bacteria are ubiquitous in disease endemic areas,Citation2 the route of transmission is largely unknown. In Australia, infection following contamination of a golf course irrigation system was reported,Citation3 while many cases elsewhere are related to disruption of the environment, e.g., due to deforestation and building of dams.Citation4 Possible sources of infection include aquatic insects, mosquitoes and mammals.Citation2,Citation5,Citation6 Person-to-person transmission is extremely rare.Citation4 Until the transmission route is better understood or an effective vaccine is developed, it will be difficult to prevent BU.
For this reason, the fight against BU is currently primarily waged by spreading information about the disease and by treating patients. The bacteria primarily infect the limbs where the skin is colder than on the trunk, as the optimal temperature for M. ulcerans is 30–33°C. Once infection is established, disease first appears as a painless nodule in the skin, which can be treated with antibiotics or surgical excision. Left untreated, the nodules can rapidly progress to extensive ulceration of the skin and in some cases bacteria can penetrate into the bones and cause osteomyelitis.Citation7,Citation8 The disease occurs primarily in resource-poor areas where limited access to and the cost of seeking healthcare can be prohibitive. This, combined with the disease symptoms generally being painless, causes many patients to not seek treatment until the disease has progressed to the ulcerative stage. At this point, infection is generally treated by surgical removal of infected skin, sometimes requiring transplantation of healthy skin tissue. Extensive lesions may even require amputation of the affected limb. Recurrence of disease is common, although concomitant antibiotic treatment can decrease the risk.Citation9 After the infection has been cleared, patients are often left with disfiguring scars and reduced mobility that can significantly reduce the quality of life and be a social stigma. This underscores the importance of developing an effective vaccine to prevent BU.
Immune response and virulence
The two best understood mycobacteria of clinical interest, M. tuberculosis and M. leprae, the causative agents of tuberculosis (TB) and leprosy respectively, are both intracellular pathogens. Th1-type cellular immune responses are essential for control of both infections, while humoral responses have little benefit and may even be detrimental to the host.Citation10,Citation11 While the immune response to M. ulcerans is not fully understood, Th1-type cellular immune responses appear to be important for control of M. ulcerans as well.Citation8,Citation12 There is some debate regarding the induction of Th2-type and anti-inflammatory cytokines by M. ulcerans. Gooding et al.Citation12 reported expression of IL-4 and IL-10, among other cytokines, from BU patients while Prévot et al. detected IL-10 and not IL-4.Citation13 Westenbrink et al., on the other hand, detected neither IL-4 nor IL-10.Citation14 These differences could be due to genetic differences in patients or M. ulcerans strains, as well as the severity of the lesions and differences in sample handling.
M. ulcerans is distinct from other mycobacteria in that it expresses a toxin that is the main bacterial virulence factor.Citation15,Citation16 The toxin, ML, is a polyketide-derived macrolide that locally suppresses T cell responses at non-toxic levels. The T cell suppression induced by ML is not completely understood, but it is clear that ML can alter both early signaling at the T cell receptor level by activation of the Src-family kinase Lck as well as blocking cytokine responses at a post-transcriptional level.Citation17 At higher concentrations, the toxin is cytotoxic.Citation8,Citation17-Citation19 Intradermal injection of the purified toxin into guinea pigs is sufficient to induce ulcerations similar to those seen in human lesionsCitation20 while Phillips et al. showed that patients with BU present immunosuppression that is reversed after treatment of BU.Citation21 This is presumably related to a decrease in toxin levels as bacteria are cleared. Coutanceau et al.Citation22 showed that infection of mice with wild type M. ulcerans but not with a ML deficient mup045 mutant led to inhibition of TNF-α expression, upregulation of MIP-2 chemokine, and host cell death within one day. Their results suggest that ML expression during the intracellular phase of M. ulcerans may contribute to immune evasion by provoking apoptosis of infected cells and altering the establishment of an appropriate inflammatory reaction. These results were later confirmed by Torrado et al. who reported that murine bone marrow-derived macrophages infected with ML-negative strains of M. ulcerans (avirulent) produce high amounts of TNF, while macrophages infected with ML-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively.Citation23 Following a proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, and the bacteria become extracellular.Citation24 This extracellular phase of infection means that humoral responses may be more important for protection than in the other mycobacterial infections, with obvious implications for vaccine development.
The ML toxin is poorly immunogenic in both mice and humansCitation25 and ML-specific antibodies have not been found. Antibody and T cell responses have, however, been detected against a number of M. ulcerans proteins. Some of these are unique to M. ulcerans while others share homology with other mycobacteria.Citation26-Citation28 A range of potential targets, coupled with recent improvements in the field of vaccine development, offers a wide variety of vaccine candidates and delivery techniques against M. ulcerans. Some of these are discussed below and summarized in .
Table 1. Summary table of possible vaccine approaches. Listed are the vaccine targets (toxin, bacteria or specific protein) and type, whether the vaccines are specific for M. ulcerans or cross-react with other mycobacteria. Results from experimental models and published immune responses in humans are listed, as well as whether the vaccine has been tested in clinical trials for protection against BU disease
M. ulcerans vaccine targets
Mycolactone (ML)
The ML toxin is an obvious BU vaccine candidate, as it is the primary virulence factor of M. ulcerans and is responsible for the disease manifestations of the bacterium. While a vaccine aimed at neutralizing the toxin or preventing it from binding to its target(s) would not prevent colonization of the bacterium, it could most likely prevent the pathological symptoms. The toxin, however, is a lipid and is poorly immunogenic in both mice and humans,Citation29 and ML-specific antibodies have not yet been found. Attempts to improve the immunogenicity of the toxin by conjugating it to a protein-carrier have had limited success (Gerd Pluschke, personal communication). Thus, a vaccine targeting the toxin is technologically challenging and unlikely to be developed in the near future.
However, the toxin is synthesized by a giant type I polyketide synthase (PKS) enzyme complex that is unique to M. ulcerans. The PKS consists of repeats of 12 enzymatic domains that serially modify and elongate the toxin precursor molecule. The precursor parts are then assembled to form a mature toxin.Citation30 Random transposon mutagenesis has also revealed other genes necessary for toxin production.Citation30,Citation31 Pidot et al.Citation28 have shown that BU patients and persons that have been exposed to M. ulcerans, but who remain disease free, can make antibodies against several of the PKS enzymatic domains. On the other hand, it remains to be demonstrated if antibodies can access the synthases in bacteria to prevent toxin production and if this can prevent BU. Alternatively, induction of synthase-specific T cell responses may help eliminate M. ulcerans-infected cells, thus improving bacterial control. To our knowledge, these synthase-specific T cell responses have thus far not been analyzed. In collaboration with Timothy Stinear, we have demonstrated that plasmid DNA vaccination can be used to analyze the immunogenicity and vaccine potential of some of these synthase domains in an experimental mouse model (unpublished data). In addition to being an exciting vaccine strategy, induction of antibody- and T cell responses against ML or its synthases might answer some fundamental questions, such as whether antibodies can penetrate the bacterium as well as the relative importance of cellular vs. humoral responses in controlling the infection. Furthermore, as ML and its synthases are specific for M. ulcerans, immunoassays based on them may be developed to improve M. ulcerans diagnosis in patients.
Live, attenuated bacteria
BCG
Bacille Calmette-Guérin (BCG) is a live, attenuated strain of M. bovis, used as a vaccine against TB, that induces significant cross-reactive immune responses against other mycobacteria. As the bacteria are alive, they can replicate in the host, inducing strong immune responses, while the attenuation ensures that they do not cause disease.Citation32 As of now, BCG is the only available vaccine against M. tuberculosis, although several new TB vaccines are in clinical or preclinical trials. These vaccines are generally used to increase or boost immune responses induced by BCG. This is achieved e.g., through use of recombinant BCG overexpressing protective antigensCitation33,Citation34 or through boosting with adjuvanted sub-unit protein vaccinesCitation35-Citation37 or viral vectors expressing M. tuberculosis antigens.Citation38
The BCG vaccine has been administered in over four billion doses, and is routinely given to newborns in developing countries because of the protection it confers against the childhood forms of TB (meningitis, miliary TB). However, the vaccine does not prevent M. tuberculosis infection and its protection against the classical, pulmonary form of TB is variable.Citation39 Most BU patients were vaccinated with BCG in infancy, which indicates that protection against BU is limited. This is supported by the results of two large clinical trials in Uganda, involving approximately 2500 and 9000 people, respectively, where the effect of BCG vaccination against BU was examined. The results showed that BCG confers transient protection against BU in 47% of vaccinated persons that can last for up to a yearCitation40,Citation41 and can shorten the duration of ulcers in patients.Citation42 Conversely, protection of children and adults from developing osteomyelitis, the most severe form of BU, is more sustained following vaccination with the BCG vaccine.Citation43
One theory to explain this is that the limited protection of BCG is due to antigenic differences between BCG and the disease-causing mycobacterial strains. This is supported by the finding that recombinant BCG expressing M. tuberculosis antigens confers greater protection than standard BCG upon challenge with M. tuberculosis.Citation44 A similar strategy of developing a recombinant BCG strain expressing immunodominant, protective M.ulcerans antigens could be considered to increase BCG-mediated protection against BU.
A booster vaccination with BCG can increase protective efficacy against leprosy but not against TB.Citation45 More recently, the BCG-REVAC cluster-randomized trial in Brazil has confirmed that revaccination with BCG does not increase its protective efficacy against TB, particularly not in regions with high incidence of nontuberculous mycobacteria.Citation46 In experimental animal models, decreased survival of guinea pigs infected with Mycobacterium tuberculosis was reported after multiple BCG vaccinations.Citation47 Finally, in an experimental mouse model, a booster vaccination with BCG did not improve the protective efficacy against BU.Citation48 Therefore, multiple immunizations with BCG cannot really be considered a rational approach for the prevention of BU.
ML-deficient M. ulcerans
An alternative BU vaccine strategy would be to use a live, attenuated ML-deficient M. ulcerans strain. The advantage of this approach is that the bacteria express all antigens expressed by virulent M. ulcerans bacteria, except for the ML toxin. Therefore, they can induce a broad spectrum of humoral and cellular M. ulcerans-specific immune responses. Several ML-deficient M. ulcerans strains exist, generated either spontaneously or through random transposon mutagenesis,Citation30 and are easily identified as they lack the characteristic yellow pigmentation of wild-type strains. Random transposon mutagenesis has revealed several genes that are involved in ML-synthesis.Citation30,Citation49 Mutation of some of these genes ablates toxin production completely, while strains with mutations in other genes can still make partial toxin.Citation49 Careful comparison of mutant strains will be necessary to identify strains that are avirulent, while still conferring significant protection against challenge with virulent M. ulcerans.
It has been recommended that at least two targeted gene deletions be introduced into live attenuated mycobacterial vaccines to increase their safety in immunocompromised individuals.Citation50 While this may negatively affect the persistence and immunogenicity of the bacteria by over-attenuating the vaccine strain, it is a necessary safety precaution as the bacteria are alive and could potentially revert to a virulent state. On the other hand, billions of doses of BCG have been administered with minimal side-effects, showing that live, attenuated mycobacteria can constitute a safe vaccine.
Sub-unit M. ulcerans vaccines
The complete sequence of the M. ulcerans plasmid and genome are publicly available (genolist.pasteur.fr/BuruList/). With this information, many M. ulcerans-specific proteins have recently been identified and shown to be immunogenic.Citation28 In addition, there are many proteins with strong homology to other mycobacterial proteins, such as antigen 85A (Ag85A) that shows an 84% amino acid sequence identity to Ag85A from M. tuberculosis.Citation26 Indeed, Tanghe et al. showed that a DNA vaccine coding for Ag85A from either M. tuberculosis or M. ulcerans was able to delay BU progression in mice, with DNA encoding Ag85A from M. ulcerans giving significantly more protection than DNA encoding Ag85A from M. tuberculosis.Citation51 In addition, Coutanceau et al. showed that DNA vaccination with M. ulcerans heat shock protein 65 conferred some protection against M. ulcerans in mice.Citation52 However, while this heat shock protein is immunogenic, it shares strong homology with human heat shock protein 60, which is a serious drawback for vaccine development.Citation53
The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of sub-unit vaccines and immunodiagnostic tests. Unfortunately, the complete genome sequence revealed the absence of these coding sequences in M. ulcerans. It has been suggested that loss of these immunodominant proteins helps the bacteria bypass the host's immunological response and that this may represent part of an ongoing adaptation of M. ulcerans to survival in host environments that are screened by immunological defense mechanisms.Citation54 However, a number of other protective antigens have been identified for M. tuberculosis (such as the -already mentioned- mycolyl-transferases of the Ag85 complex, the so-called latency antigens encoded by the DosR dormancy regulonCitation55 and members of the PPE family) and the orthologs of these antigens encoded by M. ulcerans are interesting vaccine candidates.Citation56
Carrier proteins
Attempts to induce immune responses to the ML toxin by conjugating it to protein carriers have met with limited success (discussed above). However, data published by Marsollier et al.Citation57 indicate that immune responses to insect salival proteins can confer protection against M. ulcerans when mice are subsequently exposed to M. ulcerans coated with insect salival proteins. The data further indicate that this may also apply to humans working in environments inhabited by aquatic insects. Although the mechanism of protection is not clear, it could be through coupling of M. ulcerans antigens to salival antigens that are strongly immunogenic.
While infection may be transmitted by aquatic insect bites, this does not appear to be the sole route of infection.Citation58 For other routes of infection, other antigenic proteins may be required to enhance the immune responses to M. ulcerans. BU vaccines may thus need to be customized to different regions, based on the predominant transmission route of the pathogen.
Vaccination methods
As mentioned above, a vaccine using live, attenuated bacteria is a well-established method that can induce broad immune responses against multiple antigens. For ML-deficient M. ulcerans vaccines, the only bacterial component lacking is the toxin. It is therefore possible that the vaccine-induced immune response can control the infection before a significant amount of toxin is produced. Little is known, however, about the kinetics of toxin production in humans, because of the lack of detection reagents. The poor immunogenicity of the toxin also means that it is an unlikely vaccine candidate.
DNA vaccination is a promising vaccination strategy, as M. ulcerans vaccine target genes can easily be cloned into DNA vaccination vectors and the vaccines produced in large scale. This is an inexpensive vaccine that preserves well and does not need cold storage, a major drawback with a live, attenuated vaccine. The antigens are transiently expressed by the host and DNA vaccines can induce strong humoral responses as well as both CD4 and CD8 T cell responses. Recent advances in adjuvant development have greatly improved the efficacy of this vaccination method.Citation59 DNA vaccination protocols can be improved by heterologous boosting, e.g., with a protein antigen.Citation51 Recombinant proteins can also be used to induce both humoral responses and CD4 T cell responses. However, a protein vaccine is much less efficient at priming CD8 T cells as DNA vaccines. A limitation of both DNA- and protein vaccines is that immune responses are only induced against the antigen(s) in the vaccine, and thus the breadth of the immune response is very limited. On the other hand, these sub-unit vaccines do not induce vector immunity (as is the case for recombinant viral and bacterial vectors) and they can be administered repeatedly in booster immunizations. While DNA vaccination with Ag85A has shown some protection against M. ulcerans in mice,Citation51 a sole antigen that is readily accessible to the immune response and sufficient to prevent BU has not yet been identified.
Conclusions
A great deal has been learnt about M. ulcerans and BU in the last few years.Citation1,Citation8,Citation24,Citation30,Citation60 This has led to improved therapy,Citation61,Citation62 as well as identification of possible candidates for vaccination and diagnosis.Citation26,Citation28,Citation51 However, many questions remain to be answered, such as the relative importance of humoral and cellular immune responses in controlling the infection. Both responses most likely contribute to control of infection, although their relative role may depend on the stage of infection.
Other questions, such as what the ML toxin target(s) is and the complete mechanism of toxin immunosuppression, are important, as the answers can help us gain insight into the disease mechanisms of the bacterium. Furthermore, it is possible that ML-mediated immunosuppression may decrease the efficacy of a vaccine-induced T cell response upon exposure to virulent M. ulcerans, if sufficient toxin accumulates. To study this we need a better understanding of the kinetics and in vivo conditions under which the toxin is expressed, for which we currently lack the immunological tools.
Despite technological challenges and a plethora of unanswered questions, the prospects for a BU vaccine are good. Recent work has shown that people can be exposed to and develop immune responses against M. ulcerans without developing disease symptoms.Citation28 Furthermore, people can spontaneously heal lesions without treatment, although a deforming scar can remain.Citation63 This indicates that an adaptive immune response, whether it is humoral, cellular or both, is able to control and clear the infection. However, it has not been reported whether patients who spontaneously heal lesions are left with protective memory responses, or whether they are still susceptible to re-infection. Analysis of samples from these individuals and immunological follow-up studies may help to identify correlates of protection and explain why some people develop disease while others do not.
| Abbreviations: | ||
| Buruli ulcer | = | BU |
| Mycolactone | = | ML |
| Mycobacterium ulcerans | = | M. ulcerans |
| Tuberculosis | = | TB |
| Polyketide synthase | = | PKS |
| Bacille Calmette-Guérin | = | BCG |
| Antigen 85A | = | Ag85A |
Acknowledgments
This work was partially supported by funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement N° 241500.
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