Inhibition of the Multiplication of Mycobacterium leprae by Vaccination with a Recombinant M. bovis BCG Strain That Secretes Major Membrane Protein II in Mice

The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-γ) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8⁺ T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-γ on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.Leprosy, which is caused by Mycobacterium leprae, is an infectious disease that still affects thousands of people worldwide. According to WHO''s weekly epidemiological report, 254,525 new cases were detected in 2007 (25). One reason why leprosy is still prevalent may be due to the inherent characteristics of M. leprae, i.e., slow growth and weak pathogenicity. It takes 12 to 14 days for M. leprae to replicate, so it is predicted that 2 to 5 years are necessary for the clinical manifestations to appear after an infection (1, 18). Likewise, it takes 6 to 8 months for the recognizable swelling of the footpad to appear in nude mice (22).Leprosy is clinically divided into two major categories: multibacillary (MB) leprosy and paucibacillary (PB) leprosy. In the lesions of patients with PB leprosy, dendritic cells (DCs) and activated T cells are involved with confining M. leprae to a localized area. These pathological observations indicate that cell-mediated reactions are triggered and that the activation of both CD4⁺ and CD8⁺ T cells is closely associated with inhibition of the spread of the bacilli. In contrast, abundant foamy macrophages loaded with bacilli but not DCs appear in the lesions of MB patients (11). It can be speculated that antigen (Ag)-presenting cells such as DCs recognize the immunodominant Ags of M. leprae and express those derivatives on their surfaces, thereby activating T cells. Previously, using T cells from patients with PB leprosy, we have identified major membrane protein II (MMP-II), also known as bacterioferritin (ML2038), as one of the immunodominant Ags (8). We found that MMP-II activates DCs through Toll-like receptor 2, leading to higher levels of expression of major histocompatibility complex class I and class II, CD86, and CD83 Ags and increased levels of production of interleukin-12 p70. Furthermore, MMP-II-pulsed DCs derived from patients with PB leprosy activated both autologous CD4⁺ T cells and CD8⁺ T cells to produce gamma interferon (IFN-γ) in amounts larger than the amounts produced by T cells from patients with MB leprosy and M. bovis BCG-vaccinated healthy individuals, indicating that T cells from patients with PB leprosy may be primed with MMP-II in vivo.The BCG vaccine has been used for the prevention of tuberculosis, although its role in the prevention of leprosy is still being debated. The protective efficacy of BCG against leprosy has been tested in several trials, including studies in the Karonga District of northern Malawi, in which 50% protection was observed (17). Through combined systematic analyses of experimental studies, Setia et al. found that the BCG vaccine had an overall level of protective efficacy of 26% against human leprosy (19). Their observational studies overestimated the protective effect at 61%. In another review of 29 studies, Zodpey reported that 44.8% of the reports indicated that the BCG vaccine had a level of efficacy of 50% or more (26). These observations indicate that improvements to the BCG vaccine are necessary to increase its protective effect. Recently, we produced a recombinant BCG strain that secretes MMP-II (strain BCG-SM, where SM indicates secreting MMP-II). Since MMP-II has the ability to ligate Toll-like receptor 2, we expected BCG-SM to highly activate human T cells. In fact, BCG-SM activated not only naïve CD4⁺ T cells but also naïve CD8⁺ T cells through DCs (9). The fact that BCG-SM was more efficient than the parental BCG strain at the activation of both subsets of naïve T cells led us to seek further insights into the protective activity of BCG-SM. In the present study, we investigated the effect of vaccination of BCG-SM on the multiplication of M. leprae in mice.