Jeffrey L. Fox is the ASM News Current Topics and Features Editor.
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More than Drugs Needed To Eradicate Leprosy
Investigators seek a vaccine, better diagnostic tools, and a deeper understanding of the mysteries of this obdurate pathogen
Jeffrey L. Fox
The worldwide campaign against leprosy took a great leap forward nearly two decades ago when a multidrug therapy (MDT) strategy for treating infected individuals was introduced, soon reducing the global prevalence of this disease by 85%. But, depending on how estimates are made, the leprosy case rate still hovers near 1.4 per 10,000 on a global basis. Thus, despite the remarkable success of the MDT strategy, some 1 million individuals are afflicted with leprosy, with most of them concentrated in India and, to lesser extents, Brazil, Indonesia, Bangladesh, and perhaps a dozen or more additional countries.
A more aggressive MDT campaign doubtless can reduce that global caseload still further and might even bring it in line with the World Health Organization (WHO) goal of 1.0 cases of leprosy per 10,000 by 2005. However, attaining this public health goal, laudable though it may be, falls short of eradicating the disease. Moreover, some skeptics within the community of dedicated leprosy researchers contend that a drug-based strategy, no matter how effective it now appears, is destined to stumble because the development of drug resistance is considered inevitable. And there are more mundane obstacles to its success, such as the difficulties that arise when delivering drug cocktails to impoverished patients whose broader medical needs are poorly served, if at all.
But sitting atop these seemingly straightforward obstacles to the long-term success of the MDT-based campaign against leprosy is a range of crucial microbiological subtleties that attend this relatively rare, but still much-dreaded infectious disease. Confronting those subtleties will require delving more deeply into the manifold peculiarities of Mycobacterium leprae, the pathogen responsible for causing this disease. Those challenges include its listless and—to researchers trying to grow, work with, or measure it—excruciatingly frustrating lifestyle; its richly exotic capacity for making lipids while apparently stinting on the metabolic energy stores that are required for surviving, let alone generating and decorating these ornate but characteristic fatty molecules; and its equally odd but remarkably persistent, icky, and undeniably effective means for causing a devastating disease.
M. leprae, a Clunky Bacterium but an Effective Pathogen
M. leprae—like several other members of the genus Mycobacteria—offers an initial impression of being maladapted to life as a pathogen or, for that matter, life at all. Agonizingly slow to grow in animals and still impossible to grow in culture, these bacteria seem ill-equipped to cause much damage in humans. Indeed, some striking facts back that impression. Sometimes, for example, M. leprae causes infections among humans that are so subtle as to come and go without any outward sign of disease. In other cases, symptoms may be delayed for years or even decades—adding to the challenge of monitoring this pathogen for public health purposes or for treating individual cases, while also leaving unanswered questions about how infections arise, how these pathogens withstand host immune responses in some cases but not in others, or where they reside when they are not invading the skin and nerve tissues of their unwelcoming human hosts.
Yet, despite its many clunky qualities as a bacterium, M. leprae is an extraordinarily adept, highly effective, albeit also very specialized pathogen. Thus, it is capable of dodging host immune responses while lodging within macrophages of the immune system, of residing also among assorted other cells and tissues along the outer surfaces of its human hosts where temperatures are apt to be comfortably lower for this coolness-preferring microbe, and of invading Schwann cells of the peripheral nervous system, in which it does its greatest harm by irreversibly damaging some of those nerves.
With these and other M. leprae mysteries in mind, microbiologists, infectious disease experts and mycobacteria specialists, and officials interested in keeping current modestly sized leprosy research programs on track have convened several small-scale workshops during the past few years to evaluate those programs and to develop a better sense of where they are headed. In particular, the Heiser Program for Research in Leprosy and Tuberculosis along with the National Institute of Allergy and Infectious Diseases (NIAID) sponsored one such workshop in Washington, D.C., late in 1999, and the L'Association Raoul Follereau sponsored a similar meeting in Paris last June. (Final reports from those meetings will soon be made public. For a simplified version of the principal recommendations and conclusions from the NIAID-Heiser workshop, see box, p. 152.)
In both cases, participants came away convinced that the M. leprae logjam of microbiological and immunological enigmas may be ready to break—not so much because the microorganism itself has become more tractable but more because there are new ways for working around its perverse biology. Importantly, these research efforts need to expand if the disease is ever to be eradicated, argues Roy Curtiss III of Washington University in St. Louis, Mo., who chaired the NIAID-Heiser workshop on this subject. Despite straitened research budgets, he says, "good stuff" continues to come from the few investigators who have persisted in studying M. leprae with the aim of better understanding its pathogenic interactions with humans.
Meanwhile, although improved drug regimens for treating leprosy have made some impressive inroads against the disease, that strategy for eradicating it is proving to be a "dream" based on "misplaced confidence," Curtiss says. "We need to know more about this pathogen, and we need to have smart people working on it." Eventually, some form of vaccine will be needed if that eradication goal is to be met, he adds, although designing and then delivering such a vaccine loom as major challenges.
Misunderstandings of M. leprae Biology Are Widespread
"There are a lot of misunderstandings about M. leprae," says James Krahenbuhl of the GW Long Hansen's Disease Center in Baton Rouge, La., an understatement with broad applicability, but one that undoubtedly pertains to mainstream microbiologists almost as much as it does to the general public. This pathogen "hates 37° C, it's fastidious, it cannot be cultured, and requires specialized services and committed researchers," he says. Moreover, despite some striking similarities with Mycobacterium tuberculosis and tuberculosis, on balance, M. leprae is a distinctive microbe and gives rise to a very different disease. More to the point, one can hardly exaggerate how much more challenging it can be to work with this microbe compared to M. tuberculosis, and how problematic it can be to produce adequate materials for basic and applied research on leprosy, according to both Krahenbuhl and his colleague Thomas Gillis.
The two of these M. leprae specialists devote considerable attention to generating live bacteria or inactivated cells and materials from this pathogen for other investigators to use. The major source is M. leprae-infected armadillos produced by Krahenbuhl, his colleague Richard Truman, and Patrick Brennan at Colorado State University under a National Institutes of Health contract. The other principal source of M. leprae is the footpads of mice—now usually nude mice whose immune systems are impaired—into which the mycobacteria are inoculated. Armadillos, although a productive source of M. leprae materials, typically yield a mix of viable and nonviable bacteria, whereas the mice provide the "hottest, viable bugs" at a much faster clip, according to Krahenbuhl. Meanwhile, alternative approaches to producing the bacteria in tissue culture are not so successful—in part because the bacteria thrive in cooler temperatures than the host mammalian cells can readily tolerate. Although M. leprae do not kill such cultured host cells and may elongate within them, the bacterial replication cycle is so slow that the microorganisms fail to multiply even if they seem to thrive in these cells.
Hence the necessity of producing and, to some extent, studying M. leprae in mice and armadillos. However, neither of these animals experiences a course of infection that altogether parallels leprosy in humans. For instance, although armadillos can be naturally infected, their low body temperature permits systemic and multiorgan infections that lead to death within a two-year period. This pattern is very unlike what happens in clinical cases of leprosy where the normally higher body temperature of human hosts helps to confine the infection to tissues near the body surface—typically the skin, peripheral nerves, nose, and mucous membranes.
Infections in the armadillo may even capture the two-state course in humans—"tuberculoid," in which low numbers of bacilli are observed in skin, and "lepromatous," in which high numbers of bacilli and lesions are accompanied by the absence of cellular immunity against the pathogen. However, armadillos are not congenial fixtures in animal care facilities, little is known about their genetics or immune systems, and it would be costly to develop the reagents to overcome these biological deficits.
Nude mice are stalwarts for producing M. leprae, as their feet quickly "grow to the size of your thumb" after being inoculated with the bacteria, and individuals may yield 1010 cells within 9-12 months, Gillis says. However, the immune responses of these extraordinarily immune-impaired mice are not particularly good for modeling the course of leprosy in humans. Nonetheless, systematic experiments involving a series of knock-out mouse types, each of which is missing specific immune system components such as individual cytokines, could begin to define the roles each of them play as they respond to M. leprae infections, providing an unconventional way to model these critical elements of the disease, he points out.
For example, mutant mice that are missing an inducible nitric oxide response, which is part of the normal inflammatory response of macrophage cells to pathogen-associated injuries, surprisingly do not succumb to M. leprae, Krahenbuhl says. Infecting the same genetically modified mice with M. tuberculosis would lead to "dead mice but with M. leprae there are granulomas, so we can explore this difference." Similarly, M. leprae infections are not lethal to mutant mice that no longer produce the cytokine gamma-interferon. A series of such studies can help to delineate "what kills M. leprae and which antigens stimulate specific responses," Gillis adds. "So we can do modeling in that sense. But these are very expensive experiments and require quite an investment."
Genomics Data Promise Many Insights
The students of M. leprae face plenty of challenges confronting this microbe, even without resorting to imperfect models of pathogenesis in animals such as mutant mice or balky armadillos. Until recently, it was nearly impossible to examine either the genetics or the physiology of this pathogen, according to William Jacobs, Jr., of Albert Einstein College of Medicine in Bronx, N.Y., who studies several microbes within the Mycobacterium family.
However, genomic studies are now providing complete information about the DNA sequence of both M. leprae and M. tuberculosis, making "this an unprecedented and exciting time to do research on these bugs," Jacobs says. For example, although their findings are not yet published, the sequence analysis of the M. leprae genome was completed in 2000 by Stewart Cole of the Institut Pasteur in Paris, France, and collaborators at the Sanger Centre Wellcome Trust Genome Campus in Hinxton, United Kingdom. In mid 1998, Cole, collaborators at the Sanger Centre, at NIAID, and at the Technical University of Denmark in Lyngby, Denmark reported the M. tuberculosis genome sequence.
The initial findings indicate that the M. leprae genome contains 3,268,203 base pairs and 1,700 open reading frames, making it considerably smaller and less dense in terms of apparent functional gene density than that of M. tuberculosis, which contains more than 4.4 million basepairs and 4,000 open reading frames. M. leprae appears to contain only 80 genes that are not found within the M. tuberculosis genome, and this set of genes is being scrutinized as potential markers for M. leprae infections and for clues as to its patterns of pathogenesis.
The M. leprae genome appears to contain a surprisingly high concentration—nearly half of its genome—of noncoding or pseudogenes, yet with very few apparent differences among M. leprae strains. This high degree of genomic fidelity is consistent with M. leprae being capable of scrupulously repairing DNA damage that it incurs when dealing with host defense responses or other environmental insults. This extraordinarily high fidelity plus a shortage of genes that other microorganisms carry for use in producing energy and many building blocks for growth (such as genes encoding enzymes needed for NADH metabolism) may also help to explain why M. leprae cells are so slow to replicate.
Data from such genomic sequencing and annotation efforts are and will continue to be "a tremendous help, but we've got to do genetics—to make well-defined mutants," Jacobs says. "There's no way to scan the genomic sequence and figure out all the targets and understand phenotypes."
Adaptations of More Conventional Approaches Also Provide Insights
Jacobs calls M. tuberculosis the "world's most successful pathogen, having infected one in three people on the planet." Although M. leprae is nowhere near so successful as M. tuberculosis, it, too, "can persist because it can hide from the immune response," he says. For instance, like M. tuberculosis, the M. leprae genome encodes one or several enzymes that decorate its unusual long-chain fatty acids with cyclo-propyl groups—one of probably many properties that help to confer virulence because of how they affect the host immune response to these pathogens.
Even though strictly conventional microbiological approaches to studying M. leprae remain stymied because it cannot be grown in culture, there are ways to work around some of the more obdurate features of this microorganism and perhaps to unveil some of its perplexing physiologic mysteries. For example, at first with the use of specialized phage and, subsequently, also with access to a readily transformed mutant of Mycobacterium smegmatis, M. leprae genes can be transferred into this alternative mycobacterium, which grows about 10-fold faster than M. tuberculosis, according to Jacobs.
Thus, M. smegmatis can serve as a "surrogate host" for analyzing the function of M. leprae genes. Indeed, the faster-growing mycobacterium already has played this role for the analysis of M. tuberculosis genes, including several that serve as targets for antibiotics. Moreover, phage can be used to carry genes from other mycobacteria into M. leprae as a way of determining what sorts of genes might accelerate its notoriously sluggish pace of growth.
Other approaches, including those taken by neuroscientists, also are providing insights into how this microorganism damages specialized cells and tissues of its human host. For instance, a specific component on the surface of M. leprae allows it to attack peripheral nerves, according to Anura Rambukkana at Rockefeller University in New York, N.Y., and his collaborators there, at nearby New York University Medical Center, Colorado State University in Fort Collins, and at the Max-Plank-Institut for Biochemistry in Germany. Schwann cells encase such nerve fibers and wrap around their axons to form the myelin sheath. M. leprae is the only known human bacterial pathogen that attacks the Schwann cell of the peripheral nervous system, and the nerve damage it induces is by far one of the leading causes of peripheral nerve disease in the world.
Several years ago, Rambukkana and his collaborators learned that a major component in the Schwann cell basal lamina, called lamina-2, and its receptor, called dystroglycan, are involved in M. leprae's interaction with Schwann cells. Now they find that an M. leprae-specific cell wall glycolipid called phenolic glycolipid-1 (PGL-1) binds specifically to native laminin-2, but not to other proteins in the basal lamina of the Schwann cell-axon unit. Moreover, the neural target, not the invading microbe, ultimately controls invasion of the Schwann cell. Thus, the basal lamina, a dynamic action zone, and Schwann cells respond to PGL-1 upon contact with the bacterium—opening the pathway and allowing it to enter. PGL-1 is a remarkable molecule discovered in the early 1980s by Brennan and colleagues and shown to be present in large amounts on the surface of the bacterium and to contain a specific glycolipid such that nowadays it is also used for the serodiagnosis of leprosy.
"We think clarifying PGL-1's role in nerve infection will eventually make it possible to develop strategies to block bacterial invasion of the peripheral nerve cells at an early stage and thus prevent neurological damage before the immune system gets involved," Rambukkana says. The finding could also shed light on the early stages of nerve damage in other neurodegenerative diseases such as Guillain-Barre syndrome and multiple sclerosis.
Better Diagnostic Tools and a Vaccine also Sought
The search for better diagnostic tools for leprosy to some extent reflects peculiarities of the way M. leprae infects and causes disease in humans. Although diagnosis is based mainly on clinical signs, infection often precedes telltale symptoms by many years—delaying and undoubtedly undermining the ability of drug therapy to prevent nerve damage. Moreover, except for measuring family members or other close contacts of infected individuals in the case of the PGL-1- based serology, the unavailability of reliable diagnostic tools for the early stages of infection greatly hampers efforts to screen populations at risk for this disease. Yet there is hope. The Colorado State group, in conjunction with Anandaban Leprosy Hospital in Nepal, are now conducting Phase II skin test trials in Katmandu on a new PPD-like skin test antigen.
Although identifying suitable new diagnostic tools likely will prove challenging enough, no one needs to be persuaded that such an effort should be pursued. The challenge of developing a vaccine to protect against M. leprae infections, however, falls into a somewhat different category. Thus, the success of the multidrug regimen and the current low prevalence of the disease put a twist into conventional vaccine development planning efforts.
The current assumption is that a stand-alone vaccine simply would have little or no use, except perhaps in highly endemic regions. Even that use would be tricky because of all the usual economic constraints that go with developing a vaccine for such a rare disease, for which poverty itself is widely recognized as an important risk factor. In other words, if resources could be marshaled to develop, produce, and administer a stand-alone vaccine against leprosy, the target population might by then be prosperous enough not to be at such a high risk for developing the disease and not in much need of such a vaccine.
Not so, says Marcus Horwitz at the University of California, Los Angeles, School of Medicine in Los Angeles, Calif. "Like any vaccine, a leprosy vaccine would be used for high risk groups, mainly in India, Brazil and other countries where incidence rates are high and not decreasing." Here again, although deployment of multidrug therapy is helping to reduce the prevalence of active leprosy cases in such areas, new infections continue to occur—in part because of difficulties in detecting infections at an early enough stage to prevent transmission from occurring.
Thus, he and others believe that a vaccine is needed to eradicate leprosy, and they generally agree that some kind of combination vaccine should be developed, probably one that targets both M. leprae and M. tuberculosis, if not other pathogens as well. But at this point, other controversies come into play, in part reflecting continuing frustrations over M. tuberculosis-associated vaccines, particularly the almost century-old BCG, which is an attenuated M. bovis preparation that is widely used in several, slightly different forms. M. bovis is closely related to M. tuberculosis. BCG is about 50% effective in preventing tuberculosis, and a clinical trial in India nearly a decade ago indicated that BCG by itself and also when combined with extracts from killed M. leprae cells can be partially protective against leprosy.
Another promising BCG-related approach entails modifying this vaccine through recombinant DNA techniques to make it overproduce specific secretory proteins of M. tuberculosis. and other pathogenic mycobacteria, according to Horwitz. "I was pessimistic about leprosy vaccines before when we were trying to make subunit vaccines," he says. However, overproducing these secretory proteins through recombinant techniques appears to be "key" to enhanced activity, partly because those proteins are important factors recognized by the host immune system and also because they appear to be capable of eliciting protective immunity. "The recombinant BCG vaccine expressing the major M. tuberculosis secretory protein is much more potent than BCG in protecting guinea pigs against TB," he says. "We're pursuing the same strategy with leprosy, and I suspect the TB vaccine itself may protect against leprosy because the secretory proteins [in the two mycobacteria] overlap quite a bit."
Although BCG or some modified form has proponents among would-be leprosy (and tuberculosis) vaccine developers, it also draws criticism. "M. tuberculosis knows how to subvert the immune system, so a live, attenuated vaccine like BCG is horrible in my opinion," says Washington University's Curtiss. "These mycobacterium pathogens can manipulate host responses—they are bloody smart, for crying out loud." Although he agrees that a combination leprosy-tuberculosis vaccine will be needed, he urges that its pursuit be based on more narrowly defined protective antigens through current genomics-analysis efforts and then packaging them in a combination-delivery system or perhaps in a naked DNA vaccine. "If we can figure out what the appropriate protective antigens are, that will probably lead to success," he says.
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