Find more content written by:  Maeya Ngamying; Louis Levy
  • Volume 67 , Number 3
  • Page: 302–5

Vaccination of mice against the leprosy bacillus with skin-test antigens

Maeya Ngamying; Louis Levy

To the Editor:

The specter of approximately 700,000 new leprosy cases a year, despite a prevalence of only about 805,000 (11,12), haunts us. Many, perhaps the majority, of these cases are detected in the course of intensive case finding (11), and are single-lesion cases which are difficult to diagnose definitively. Measurement of the true incidence of leprosy requires new diagnostic tools, more sensitive than physical examination supported at times by slit-skin smear (9). Neither serology, even the modern, user- friendly, sensitive phenolic glycolipid-I (PGL-I)-based kits (5), nor gene-amplification based on a variety of primers provides the necessary sensitivity (10). A promising approach to the diagnosis of asymptomatic leprosy is provided by the availability of two new skin-test antigens-MLSA-LAM (soluble antigens of Mycobacterium leprae from which the immunosuppressive lipoarabinomannan has been removed) and MLCwA ( M. leprae cell-wall antigens) (2). These materials have successfully undergone Phase I safety studies in the U.S.A., and Phase II studies arc currently being planned for several areas of high leprosy endemicity. Awaiting the outcome of these studies, we examined the protective potency of these products in the B ALB/c mouse.

M. leprae harvested from the livers and spleens of M. leprae -infected armadillos were disrupted by sonication and centrifuged to yield two major fractions-the soluble supernatant (MLSA) and the insoluble pellet (MLCwA) (2). Much of the soluble carbohydrates and lipids in the preparations, especially lipoarabinomannan (LAM) and the phosphatidylinositol mannosides, were removed by extraction with Triton X-114 to yield the two fractions, MLSA-LAM and MLCwA-LAM (1). These materials were produced at Colorado State University under the conditions required for approval of their use as skin-test antigens in humans (1). BALB/c mice were bred under specific pathogen-free (SPF) conditions in the animal facility of the Sasakawa Research Building, Ministry of Public Health, Non- thaburi, Thailand, from a nucleus purchased from CLEA Japan, Inc., Kawasaki, Japan. The inoculum of M. leprae was prepared from the foot pads of BALB/c mice that had been inoculated approximately 4 months earlier with a suspension of organisms of the "Thai 53" strain that had been provided by Dr. J. L. Krahenbuhl, Baton Rouge, Louisiana, U.S.A. When the organisms were found to have multiplied to a number approaching 10" per foot pad, the bacterial suspension was diluted to a concentration of 5 x 10' organisms per 0.03 ml.

For the purpose of this experiment, the mice of one group were held as untreated controls, and those of other groups were administered incomplete Freund's adjuvant (IFA) or aluminum hydrogel (APOH), an adjuvant suitable for use in humans, or one of the two antigen preparations suspended in one of the adjuvants intracutaneously in the flank. After 1 month, the mice were challenged in the right hind foot pad with 5 x 103 M. leprae . At intervals thereafter, the organisms were harvested from the pooled hind foot pads of four control mice, stained by a standard, room temperature acid-fast stain, and counted directly (6-8).

The results of this experiment are presented in The Table as the actual number of acid-fast bacilli (AFB) per individual foot pad as well as the median number of AFB per foot pad for each group. These results demonstrate that all three antigens were protective when administered to mice after emulsification in IFA; whereas none of the three antigens protected mice when administered in APOH.

Previously, we reported that immunization of mice with various cell-wall fractions of M. leprae , progressively depleted of lipids, carbohydrates, and soluble proteins, conferred significant protection against subsequent infection with live M. leprae (3), and subsequent studies (4) demonstrated that a mixture of proteins derived from the M. leprae cell wall conferred long-term protection, compared to the shorter-term protection provided by individual proteins. The conclusion from this experiment was that multiple M. leprae protein epitopes are critical for solid vaccine protection against the infection. The activity of the fractions studied in this experiment, MLSA and MLCwA, from which LAM and other apparently immunosuppressive lipids and li- poglycans have been removed, provides additional evidence of the utility of multicom- ponent fractions as distinct from individual proteins.


- Maeya Ngamying, M.S.
Louis Levy, M.D., Ph.D.

Sasakawa Research Building
National Institute of Health
Department of Medical Science
Ministry of Public Health
Tiwanond Road
Nonthaburi 11000, Thailand

- Patrick J. Brennan, Ph.D.

Department of Microbiology
Colorado State University
Fort Collins, Colorado 80523, U.S.A.

Acknowledgment. We thank the Sasakawa Memorial Health Foundation for support of this research and Dr. Yo Yuasa for his interest and encouragement. Special appreciation is extended to scientists Ms. Phanida Phaknilrat, Ms. Sopa Srisungnam, and Ms. Sukanya Wattanapokayakit at the Sasakawa Research Building. The antigens were produced under the terms of NIH, NIAID Contract NO I AI-55262 with Colorado State University.



1. BB-IND 7938. FDA designated title Mycobacterium leprae (soluble antigen MLSA-LAM; cell wall antigen MLCwA) skin test antigens (armadillo-derived). 1998.

2. Brennan, P. J., Donahue, S. A., Scott, D. K., Rivoire, B. L. and Walsh, G. P. A plan for preparation and application to humans of new leprosy skin-test antigens. Int. J. Lepr. 63(1995)645-646.

3. Gelber, R. H., Brennan, P. J., Hunter, S. W., Mijnn, M. W., Monson, J. M., Murray, L. P., Siu, P., Tsang, M., Engleman, E. G. and Mo- hagheghpour, N. Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae . Infect. Immun. 58(1990)711-718.

4. Gelber, R. H., Mehra, V., Bloom, B., Murray, L. P., Siu, P., Tsang, M. and Brennan, P. J. Vaccination with pure Mycobacterium leprae protein inhibits M. leprae multiplication in mouse foot pads. Infect. Immun. 62(1994)4250-4255.

5. Klatser, P. R., Cho, S.-N. and Brennan, P. J. The contribution of serological tests to leprosy control. Int. J. Lepr. 64 (Suppl.) (1996) S63-S64.

6. Shepard, C. C. The experimental disease that follows the injection of human leprosy bacilli into foot pads of mice. J. Exp. Med. 112(1960)445-454.

7. Shepard, C. C., Van Landingham, R. M. and Walker, L. L. Immunity to Mycobacterium leprae infections in mice stimulated by M. leprae , BCG, and graft-versus-host reactions. Infect. Immun. 14(1976)919-928.

8. Shepard, C. C., Van Landingham, R. M., Walker, L. L. and Ye, S. Z. Comparison of the immunogenicity of vaccines prepared from viable Mycobacterium bovis BCG, heat killed Mycobacterium leprae , and a mixture of the two for normal and Mycobacterium leprae -\a\tnn\. mice. Infect. Immun. 40(1983)1096-1103.

9. Smith, W. C. S. We need to know what is happening to the incidence of leprosy. Lepr. Rev. 68(1997)195-200.

10. Williams, D. L., Gii.lis, T. P., Booth, R. J., Looker, D. and Watson, J. D. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae . J. Infect. Dis. 162(1990)193-200.

11. World Health Organization. Action Programme for the Elimination of Leprosy. Status Report 1998. WHO/LEP/98.2(1998)1-57.

12. World Health Organization. L'élimination de la lepre on tart que problème de senté publique (mise 'a jour). Wkly. Epidemiol. Ree. 73(1998)305-312.











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