Identification of Murine H2-D^d- and H2-A^b-Restricted T-Cell Epitopes on a Novel Protective Antigen, MPT51, ofMycobacterium tuberculosis

Abstract

Both CD4؉ type 1 helper T (Th1) cells and CD8 ؉ cytotoxic T lymphocytes (CTL) play pivotal roles in protection against Mycobacterium tuberculosis infection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-␥) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-␥. The world-wide problem of tuberculosis (TB) is increasing due to several factors, including multi-drug-resistant strains and coinfection with human immunodeficiency virus (32). An attenuated strain of Mycobacterium bovis BCG is the only currently available anti-TB vaccine, yet its efficacy, particularly its efficacy against pulmonary TB in adults, is controversial (42). It is evident that there is an urgent need for a new and more reliable anti-TB vaccine (23).Although the mechanisms of protection against TB have not been completely determined, cell-mediated immunity plays a pivotal role in the control of Mycobacterium tuberculosis infection. There is mounting evidence that CD4 ϩ type 1 helper T (Th1) cells are involved in the development of resistance to the disease, primarily through the production of macrophage-activating cytokines, such as gamma interferon (IFN-␥) (22). In addition, CD8 ϩ cytotoxic T lymphocytes (CTL) contribute to disease resistance since susceptibility to M. tuberculosis is greater in mice deficient in CD8 ϩ T cells (21, 41). In fact, mice with a ␤2-microglobulin deficiency are susceptible to M. tuberculosis infection (13,36,40). However, these mice are more susceptible than mice deficient in major histocompatibility complex (MHC) class Ia molecules, perhaps due to derangement of ␤2-microglobulin-dependent systems other than the MHC class Ia system, such as the iron uptake system of the host macrophages that depends on ␤2-microglobulin (38). Human CD8ϩ CTL recognize mycobacterial cell wall-derived lipid and glycolipid antigens in the context of group 1 CD1 molecules (CD1a, -b, and -c) and ␤2-microglobulin-dependent MHC class Ib molecules expressed almost exclusively on dendritic cells (34), but the in vivo function of these cells remains unknown.To design a new generation of vaccines, more information on the antigenic makeup of M. tuberculosis must be obtained in order to identify immunodominant proteins and epitopes. Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB (31). The mouse model of TB infection has revealed that memory T cells from immune mice produce a substantial amount of IFN-␥ in response to two fractions of M. tuberculosis culture filtrate represented by 6-to 10-kDa proteins and the antigen 85 (Ag85) complex, a family of 30-to 32-kDa proteins (2). The Ag85 complex (Ag85A, Ag85B, and Ag85...

Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived fromMycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

“…Peptides. Peptides spanning the entire mature MPT5l amino acid sequence of M. tuberculosis (266 amino acid residues) were synthesized as 20-mers overlapping by 10 residues; the only exception was the carboxyl-terminal 12-mer from amino acid 255 to amino acid 266, which was described previously (34). Briefly, lyophilized peptides were purchased from Invitrogen Corporation (Carlsbad, CA), and the purity was confirmed by mass spectrometry.…”

“…Supernatants were harvested 24 h later and stored at Ϫ20°C until they were assayed. The concentration of IFN-␥ in the culture supernatants was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) The ELISA was carried out as described previously (34), with some modifications. The following method was used.…”

“…Immunization of mice. Mice were immunized with pCI-MPT51, a plasmid DNA vaccine encoding the mature MPT51 molecule (34), employing a gene gun bombardment system. For DNA immunization with the Helios gene gun system (Bio-Rad Laboratories, Hercules, CA), cartridges of DNA-coated gold particles were prepared according to the manufacturer's instructions.…”

“…Another major secreted protein, MPT51, was demonstrated to cross-react with the three components of the Ag85 complex at antibody levels and to exhibit primary protein structure similarity (37 to 43% at the amino acid level) with these components (22,36). Using a DNA vaccine encoding MPT51, we found that MPT51 can induce specific cellular immune responses and protective immunity against challenge with M. tuberculosis (20), and we identified murine T-cell epitopes using C57BL/6 and BALB/c mouse strains (34).…”

Mycobacterium tuberculosis-Specific CD8+ T Cells and Their Role in Immunity

“…DNA vaccination of mice elicited CTL activity against four of the peptides. Two of the peptides (Ag85B [143][144][145][146][147][148][149][150][151][152] and Ag85B [199][200][201][202][203][204][205][206][207] ) were able to stimulate short-term antigenspecific CD8 + T cell lines from BCG-vaccinated individuals. In the case of Ag85B [199][200][201][202][203][204][205][206][207] , tetramers were made and CD8 + T cells that stained with the tetramers were enriched in the short-term cell lines.…”