Leshmaniasis
On the Trail of a Vaccine for a Stubborn Emerging Disease
Steven G. Reed
Kenneth D. StuartLeishmania are protozoan parasites that cause several types of human disease ranging from skin lesions to fatal internal illness. They are found in every continent but Australia and Antarctica, and cause approximately 15 million cases of human disease annually The number may be much higher, because infections often go undiagnosed. The parasite is transmitted by sandflies, which are found in a wide range of habitat, from desert to tropical rain forest.
Among the many varieties, or species, of Leishmania, approximately 10 are important in human disease. The most common form of infection is a severe skin lesion that can be disfiguring. In some regions of the Middle East a traditional form of vaccination involves scarifying material from an active lesion into the skin of a young child in an area of the body where the resulting scar will not be noticeable. This time-honored procedure prevents the development of further disease and illustrates the feasibility of developing an effective synthetic vaccine.
The most serious and fatal form of leishmamasis is internal, with no visible skin lesion. This form, visceral leishmaniasis, is more difficult to diagnose. Leishmania is an important opportunist in immunocompromised patients, and is an emerging disease agent, particularly in developing countries and in southern Europe. The parasite also can be carried by dogs and other mammals, from which sandflies can be infected and then pass the infection to human beings (Figure 1) . The infections are often contracted in the course of routine daily outside activity in rural or suburban locations.
Leishmanial infections are generally difficult to treat. The parasites are one of the few organisms that live inside of macrophages, a type of white blood cell of pivotal importance in the immune system. Leishmania infect and reproduce in macrophages and thereby affect normal immune responses. This fascinating adaptation to survive in such a hostile environment explains why it is so difficult to treat leishmanial infections; it is difficult to kill them without also killing host cells. The most common drugs used, such as antimonials and amphotericin, are toxic, and some patients die from these drugs.
The variety of species involved in human disease, and the wide spectrum of disease, ranging from subclinical to lethal, have made the development of diagnostic tests and vaccines especially difficult. Our work in this area has focused on detecting visceral leishmaniasis. In arid regions of Africa, India, China, and Latin America, these infections are common, particularly in children, and are often fatal. In Europe, the infections typically occur in dogs, but have recently become a common problem in persons with AIDS.
Control efforts, including insect spraying and elimination of dogs, generally have been ineffective. The leishmanial diseases continue to increase as a consequence of urbanization in developing nations. People who migrate from rural to suburban areas may carry sandfly infestations or bring infected dogs and thus create new foci of disease. This problem - combined with population growth, malnutrition, and increase in AIDS - has led the World Health Organization to predict a rapid increase in leishmaniasis in the next few years.
A Diagnostic Test for Visceral Leishmaniasis
The development of new and effective vaccines and diagnostics for leishmaniasis requires an intense international effort with close collaboration among laboratories with sophisticated research in genetic technologies and clinical centers in countries where the diseases are endemic. We have developed close ties among our laboratories in Seattle and researchers in Brazil, India, Africa, China, and the Middle East. The application of patient material, particularly sera and white blood cells, makes it possible to develop probes to screen gene libraries to develop recombinant proteins for use as diagnostic reagents or as vaccines. Such proteins will allow the widespread application of effective and standardized diagnostic tests and vaccines.In 1994, we developed a simple blood test for the rapid detection of visceral leishmaniasis. When we began this work, diagnosis was made by bone marrow biopsy Our test requires only a drop of blood and can be done in the field after brief training. The development of the test was made possible by the molecular cloning of the leishmanial K39 protein, which has a strong affinity for specific antibody Recombinant DNA technology allows production of large amounts of protein. Normal persons have no antibody to K39. The K39 test can be produced for less than $1 per test, and its sensitivity and specificity are greater than 99%. The test is now being widely used, and is having a significant effect on the treatment and control of visceral leishmaniasis.
We also applied our experience with the K39 test to a medical problem related to the Persian Gulf War of 1990-91. Hundreds of military personnel have complained of chronic illnesses that have been difficult or impossible to diagnose. We knew that many troops were deployed in areas where leishmaniasis is endemic, and researchers at the Walter Reed Army Institute of Medical Research had detected leishmania parasites in the bone marrow of several Gulf War veterans. The K39 test was not effective in these soldiers because these Leishmania were of a different species than those causing traditional visceral leishmaniasis.
Using an approach similar to that taken to develop the K39 test, we screened libraries of Leishmania genes with serum from the veterans from whom Leishmania had been isolated. We identified a previously unknown leishmanial protein that reacted with the blood from the infected veterans. The new blood test has enabled us to diagnose several persons not diagnosed by other means. It appears that "Gulf War illness" has a variety of causes, and only some of the affected military personnel have leishmaniasis.
Vaccine Research
Much of our research focuses on vaccine development. For many infectious diseases, particularly those of developing countries, vaccination remains the most feasible, cost-effective control method. We have cloned Leishmania genes that code for proteins that stimulate protective immune responses in T cells from persons who have recovered from disease and are therefore immune. Although we took advantage of strong antibody responses to develop diagnostic tests, the antibody produced during leishmaniasis is not correlated with cure or immunity. Rather, the cellular response is the protective one. Therefore, proteins that stimulate T cells will be those needed for vaccine development. Our labs have pioneered the use of human T cells for the identification of leishmanial proteins that may be vaccine candidates. Over the past 10 years, we have identified more than 12 such proteins that are now being tested as vaccines in animals. Clinical trials have begun in Brazil. We hope that an effective vaccine will be available within the next five years.
Figure 1: The vector-host cycle for several species of Leishmania.
The organisms live as flagellated, extracellular promastigotes in the digestive tract of the sandfly vector. When the sandfly bites a mammalian host such as a human, dog, or mouse, the promastigotes infect macrophages where they transform into intracellular amastigotes. These disseminate via the lymphatic and vascular systems to produce the symptoms associated with the specific species of Leishmania transmitted by the sandfly The cycle begins anew
when a sandfly takes a blood meal from an infected host and the amastigotes transform into promastigotes and then multiply in the sandfly gut.This work to develop diagnostics and vaccines relies on identification of specific genes of the parasite. In addition, the genome also encodes important traits such as drug resistance, virulence, infectivity, and host or vector range. Some traits are polygenic and some are products of single genes. For example, amplification of specific genes is responsible for resistance to various drugs or enhances the nutritional or immune escape capabilities of this pathogen. Furthermore, studies of Leishmania and related parasites have led to the discovery of important genetic regulation mechanisms such as RNA editing, transsplicing, and structure of membrane proteins. Genome Mapping
Recently, an international Leishmama Genome Network has been organized under the auspices of the World Health Organization to map the genome of this parasite. Leishmania are experimentally convenient. The complete life cycle can be maintaine parasites can be grown in the laboratory and in experimental animals, and the parasites , genes can be manipulated. The genetic content is of modest size compared to the human genome and is organized into 36 pairs of chromosomes. The Leishmania genome has relatively little noninformational or "junk" DNA, so mapping and sequence analysis is simpler than for more complex organisms. Initial analysis reveals that the genome is compact; the genes essentially abut one another.Work in many laboratories resulted in the sequencing of numerous scattered regions of the genome from several Leishmania species. Much of the sequenced DNA has been localized to specific chromosomes and used as probes to contribute to the development of a map of the entire Leishmania genome. This information has been consolidated in a database in Cambridge, England. We have sequenced about half of the smallest Leishmania chromosome and a segment that is frequently amplified (Figure 2). This work has already identified several important genes including a transporter, a protein kinase, an immune regulator, enzyme synthesizers, ribosomal protein synthesizers, subcellular localization proteins, and a growth regulator.
The Leishmania genome project may allow rapid identification of genes important for diploid eukaryotic pathogen. Ensuing studies could use this information to help eliminate this important pathogen and to learn principles of pathogenic disease strategy and aspects of macrophage function that can be applied to a variety of important diseases.
Recommended Reading
Badaro R, Benson DR, Eulalio MC, et al: rK39: A cloned antigen of Leishmania chagasi that both detects and predicts active visceral leishmaniasis. J Infect Dis 1996-1 173:758-762.
Burns JM, Shreffier WG, Benson DR, et al: Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in both African and American visceral leishmaniasis. Proc Nat Acad Sci USA 1993; 90:775-779.
Skeiky YAW, Guderian JA, Benson DR, et al: A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin-12. J Exp Med 1995; 181:1527-1537.Authors
Steven G. Reed, Ph.D., is an associate professor in the Department of Pathobiology at the UW School of Public Health and Community Medicine, and director of the Infectious Disease Research Institute and executive vice president and director of antigen discovery at Corixa Corporation, a Seattle biotechnology firm.
Kenneth D. Stuart, Ph.D., is professor and chair of the Department of Pathobiology at the UW School of Public Health and Community Medicine, and director and senior scientist at Seattle Biomedical Research Institute.
Figure 2: Regions of chromosome 1 that were sequenced
in the Leishmania genome project. Boxes indicate protein
coding regions of genes that were recognized and those
whose functions have been identified are labeled.
Arrows indicate direction of transcription.
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Created: 3/3/98 Updated: 7/15/99
