• Volume 69 , Number 1
  • Page: 44–5

Effects of vaccination with several mycobacterial proteins and lipoproteins on Mycobacterium leprae infection of the mouse

Maeya Ngamying; Paijit Varachit; Phanida Phaknilrat; B.S. Louis Levy; Patrick J. Brennan; Sang-Nae Cho

To the Editor:

Continuing our study (5) of the protective effects of various components of Mycobacterium leprae and other mycobacteria against infection of the mouse foot pad with M. leprae, we examined the potential of several proteins and lipoproteins derived from both M. leprae and M. tuberculosis to protect mice against challenge with M. leprae in the hind foot pad.

One-hundred-seventy, female BALB/c +/+ mice, purchased from CLEA Japan, Inc., Meguro-ku, Japan, and housed locally at the Sasakawa Research Building under specific-pathogen-free conditions, were divided among 11 groups as shown in Table 1. The four antigen preparations, which were generated at Colorado State University, Fort Collins, Colorado, U.S.A., or Yon- sei University, Seoul, Korea, were: M. leprae soluble antigen from which lipoarabinomannan had been removed (MLSA- LAM); a pool of recombinant M. leprae proteins [antigen 85A (32 kDa) and antigen 85C (30 kDa)]; a pool of recombinant M.tuberculosis proteins [38 kDa, antigen 85A (32 kDa), antigen 85C (30 kDa), 19 kDa, 23 kDa, and 14 kDa]; and a pool of native M. tuberculosis lipoproteins prepared by extraction of M. tuberculosis with the detergent TX114 C). These were employed with two adjuvants-Freund's incomplete adjuvant (FIA) and monophosphoryl lipid A (MPL) solubilized in triethanolamine (TeoA) (1)



The antigens, which were emulsified in one of the adjuvants in every case but one, were injected intradermally in each flank to groups of 15 mice in a dosage of 20 µg per mouse on three occasions 3 weeks apart. The pooled M. tuberculosis lipoproteins were administered to one group of mice without added adjuvant in the hope that the inherent lipid element would provide its own adjuvanticity. Additional groups of 15 mice were administered one of the adjuvants, and a final group of 20 mice, not treated, served as the controls. Twenty-eight days after the third injection, the antigen-treated mice were inoculated, each with 5 x 103 M. leprae into the right hind foot pads (RHF), and the control and adjuvant-treated mice were similarly inoculated in both hind foot pads (BHF). Harvests of M. leprae from the inoculated mice were subsequently performed by Shepard's method (6-7).

Because the organisms had not multiplied in large proportions of the control mice that had been subjected to harvest earlier than 200 days after inoculation, the results (Table 2) suggest that the inoculum had contained only a small proportion of viable M. leprae. Assuming that the organisms would have multiplied in all of the foot pads inoculated with 5 x 104 M. leprae per foot pad, and in none of those inoculated with 500 organisms per foot pad, one may calculate by means of the HalversonZiegler equation (3) that the inoculum contained only about 4 viable organisms per 5 x 104 total, i.e., fewer than I per 10,000.



Nevertheless, the outcome of this vaccine trial is clear-cut (Table 3). In interpreting this table, one must compare like with like. For instance, the results in mice treated with antigen in FIA must be compared with the results in mice administered FIA alone; similarly with MPL. Only the mice administered the native lipoproteins without adjuvant should be compared with the untreated control mice. The results of harvests performed between 210 and 231 days after inoculation from all groups of mice suggest that the mice were protected against challenge with M. leprae only by MLSA-LAM, if the antigen had been suspended in MPL. The recombinant proteins were without effect, as was the pool of native M. tuberculosis lipoproteins, which, if administered without adjuvant, appeared to enhance multiplication of the organisms.



The lack of effectiveness of the pools of recombinant proteins was disappointing, be­cause recombinant products provide the best hope for a subunit vaccine against leprosy in an era of diminishing availability of armadillo-derived M. leprae. The evidence of efficacy of whole, complex fractions of M. leprae containing a multitude of immunogenic proteins, in addition to some carbohydrates, lipoglycans, and lipids, is in accord with other successful studies (2). demonstrating that only a complex array of immunogens provides protection against leprosy.


- Maeya Ngamying, M.S.
Paijit Varachit, M.D.
Phanida Phaknilrat, B.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 110000, Thailand

- Patrick J. Brennan, Ph.D.

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

- Sang-Nae Cho, D.V.M., Ph.D.

Department of Microbiology
Yonsei University College of Medicine
Seoul 120-752, Korea

Acknowledgment. We thank ihe Sasakawa Memorial Health Foundation for support of this researeh and Dr. Yo Yuasa for his interest and encouragement. Special thanks are extended to Prof. Teera Ramasoota, Dr. Boosbun Chua-Intra, and Dr. Raywadee Butraporn. The recombinant antigens were produced with support by a grant from the Korea Science and Engineering Foundation (No. 981-0710-076-2). The remaining antigens were produced under the terms of NIH, NIAID Contract NOl AI-55262 with Colorado State University.



1. Baldwin, S. L., D'Souza, C, Roberts, A. D., Kelly, B. P., Frank, A. A., Liu, M. A., Ulmer, J. B.. Huygen, K., McMurray, D. M. and Orme, I. M. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect. Immun. 66(1998)2951-2959.

2. Gupte, M. D.. Vallishayee, R. S., Arantharaman. D. S., Nagaraju. B., Sreevata, Balasubramanyam. S., dk Britto, R. L. J., Elango, N., Uthayakumaran, N., Mahalingam, V. N., Lourdusamy, G., Ramalingam. A., Kannan. S. and Arokiswamy, J. Comparative leprosy vaccine trial in South India. Indian J. Lepr. 70 (1998) 369-388.

3. Halverson, H. O. and Ziegler, N. R. Application of statistics to problems in bacteriology. I. A means of determining bacterial population by the dilution method. J. Bacteriol. 25(1933)101-121.

4. Hunter, S. W., Rivoire, B., Mehra, V., Bloom, B. R. and Brennan. P. J. The major native proteins of the leprosy bacillus. J. Biol. Chem. 265(1990)14065-14068.

5. Ngamying, M, Levy, L. and Brennan. P. J. Vaccination of mice against the leprosy bacillus with skin-test antigens. Int. J. Lepr. 67(1999)305-307.

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

7. Shepard, C. C. and McRae, D. H. A method for counting acid-fast bacilli. Int. J. Lepr. 36(1968)78-82.











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