Laboratory Research: Pathogenesis and Vaccines
Mechanisms of nerve injury in leprosy
Investigator: David Scollard , M.D., Ph.D.
Nerve injury is the major cause of morbidity in leprosy, the major infectious cause of crippling in the world.
The mechanisms of this injury are very poorly understood: human nerves are seldom biopsied, so direct data are minimal, and there have been no animal models until the recent recognition of leprosy neuritis in the armadillo. In this animal, intravenous inoculation of M. leprae results in infection of peripheral nerves.
National Hansen's Disease Program studies of this model suggest that infection begins in epineurial lymphatics and blood vessels, from where it spreads to the well-protected endoneurial compartment. We hypothesize that endothelial cells of the peripheral nerve are the gatekeepers allowing entry of M. leprae but not other bacterial pathogens. Since reagents to study this further in the armadillo have been unavailable, we have proceeded using an in-vitro cell culture model to analyze the effects of M. leprae infection on
- the viability of endothelial cells,
- the permeability of monolayers created with these cells and
- the expression of cell surface molecules involved in cell to cell contact.
Using the armadillo model of Hansen's disease, we are now preparing for immuno-histochemical and molecular studies of armadillo nerves with varying degrees of M. leprae infection. The goal of these studies is to provide the first detailed description of the development of immunity and inflammation in infected nerves, enabling the formulation of specific hypotheses concerning the mechanisms of development and progression of lepromatous neuritis.
Endocrine Changes as Risk Factors for Leprosy Reactions
Investigator: David Scollard, M.D., Ph.D.
Much of the crippling in leprosy results from acute, immunologic "reactions," but the factors that initiate them are unknown. Longstanding clinical observations suggest that hormonal changes may be associated with reactions and are possible precipitating factors, but no good quantitative data exist.
An international, collaborative, prospective study is now underway, with a goal of enrolling approximately 2,700 new patients in three endemic areas: Manaus and Goiania in Brazil , Cebu in The Philippines, and Lalgadh , Nepal .
Levels of several hormones and cytokines will be measured in paired baseline and follow-up blood samples from selected patients, matched using a nested case-control design. Levels will be correlated with reaction status and nerve injury. This novel approach combines the preeminent endocrinology research capabilities of collaborators at the National Institutes of Health with established leprosy expertise in field sites and at the National Hansen's Disease Program.
Biological Activity of Thalidomide
Investigator: Edward Shannon, Ph.D.
Erythema nodosum leprosum (ENL) is a medical complication that can occur in multibacillary Hansen's disease patients. The treatment of choice for ENL is thalidomide.
The sequence of events that precipitate ENL, as well as the mechanism(s) by which thalidomide and/or steroids arrest ENL, have not been elucidated. The diverse effects of thalidomide on TNF-a from clinical trails, and sepsis studies in rodents may be due to many variables.
Consequently, interpretation of the literature regarding thalidomide's ability to regulate TNF-a in ENL and other inflammatory conditions is confusing.
The National Hansen's Disease Program's current research effort is to determine thalidomide's effect on IL-6 and IL-8 as well as antibody synthesis based on recent discoveries that thalidomide, in combination with dexamethasone, has a positive therapeutic effect on patients with multiple myeloma, a condition in which immunoglobulin synthesis continues unabated. If a linkage can be established between thalidomide's ability to regulate cytokines known to be important in immunoglobulin synthesis, then this approach can be used to further our understanding of potential mechanisms active in ENL.
Antigen discovery and vaccine development
Investigators: Tom Gillis, Ph.D, Jim Krahenbuhl , Ph.D., and Linda Adams, Ph.D.
Despite significant improvement in global control measures for leprosy, new cases of leprosy continue at an incidence of 500-600,000 per year. Implementation of worldwide World Health Organization-recommended multidrug therapy (WHO-MDT) has raised the hope of reducing leprosy to manageable levels but elimination of the disease is not imminent and will require other intervention strategies and tools for improving early diagnosis, minimizing transmission to individuals at risk for infection and for vaccinating against infection.
By far the intervention strategy with the greatest impact would be an effective vaccine against leprosy. Understanding resistance to leprosy and tuberculosis requires identification of antigens that stimulate cell-mediated immune responses.
The fact that M. leprae remains one of the few bacterial pathogens of humans that has not been successfully cultivated in vitro poses a serious challenge to the identification and purification of protective antigens. The National Hansen's Disease Program strategy is to test new DNA vaccines and in mice with the aim of inducing protective immunity to challenge with live M. leprae and to characterize the immune response associated with protection or absence of protection.
Our studies are exploring the possibility that DNA vaccination against leprosy may exhibit improved characteristics over conventional protein and whole bacterial vaccines and provide cross-protection against both leprosy and tuberculosis. We are also testing auxotrophic mutants of M. tuberculosis and recombinant BCG vaccines expressing M. tuberculosis or M. leprae protein antigen. We are also exploring the utility of gene knock out mice by studying their immune responses to vaccination as well as what immunological cues are necessary to produce protective immunity to M. leprae infection.
Development of a diagnostic test for preclinical leprosy
Investigator: Jim Krahenbuhl, Ph.D.
Since 1992 WHO MDT regimens have reduced clinical leprosy prevalence by >90% yet incidence remains virtually unchanged demonstrating treatment alone does not block transmission and underscoring our lack tools for detecting sub clinical leprosy to determine its true incidence / prevalence.
A major effort is underway to employ recombinant and synthetic peptides and proteins to develop diagnostic assays for sub clinical infection using in vitro T cell reactivity and a new generation of serological tests. As M. leprae can be virtually undetectable in even early clinical leprosy, CMI and antibody response in subclinical leprosy will likely be weak. Thus, the sensitivity of new diagnostic tests is as important as specificity.
The National Hansen's Disease Program will use the early phase of the mouse foot pad model of leprosy as a “preclinical” model to screen T cell and B cell reactivity of M. leprae synthetic and recombinant peptides and protein antigens provided by three collaborating laboratories. Unlike human disease, our animal model offers important advantages, e.g. we can control infection progress and monitor bacillary growth relative to time course development of T cell sensitization and antibody response to antigens already available from four collaborating labs. These animal studies will provide an important interface between antigen development and the expensive, problematic screening of these antigens in humans under field conditions.
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