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

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-γ) 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-γ. Flow cytometric analysis with intracellular IFN-γ and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8(+) T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with K(d), D(d), or L(d) indicated that the epitope is presented by D(d). Finally, we proved that the p24-32/D(d) complex is recognized by IFN-γ-producing CTL. In C57BL/6 mice, we observed H2-A(b)-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine against M. tuberculosis infection

Microfluidic-prepared DOTAP nanoparticles induce strong T-cell responses in mice.

For the induction of antigen-specific T-cell responses by vaccination, an appropriate immune adjuvant is required. Vaccine adjuvants generally provide two functions, namely, immune potentiator and delivery, and many adjuvants that can efficiently induce T-cell responses are known to have the combination of these two functions. In this study, we explored a cationic lipid DOTAP-based adjuvant. We found that the microfluidic preparation of DOTAP nanoparticles induced stronger CD4+ and CD8+ T-cell responses than liposomal DOTAP. The further addition of Type-A CpG D35 in DOTAP nanoparticles increased the induction of T-cell responses, particularly in CD4+ T cells. Further investigations revealed that the size of DOTAP nanoparticles, prepared buffer conditions, and physicochemical interaction with vaccine antigen are important factors for the efficient induction of T-cell responses with a relatively small antigen dose. These results suggested that microfluidic-prepared DOTAP nanoparticles plus D35 are a promising adjuvant for a vaccine that induces therapeutic T-cell responses for treating cancer and infectious diseases

Carbonate Apatite Nanoparticles Act as Potent Vaccine Adjuvant Delivery Vehicles by Enhancing Cytokine Production Induced by Encapsulated Cytosine-Phosphate-Guanine Oligodeoxynucleotides

Vaccine adjuvants that can induce not only antigen-specific antibody responses but also Th1-type immune responses and CD8+ cytotoxic T lymphocyte responses are needed for the development of vaccines against infectious diseases and cancer. Of many available adjuvants, oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs are the most promising for inducing the necessary immune responses, and these adjuvants are currently under clinical trials in humans. However, the development of novel delivery vehicles that enhance the adjuvant effects of CpG ODNs, subsequently increasing the production of cytokines such as type-I interferons (IFNs), is highly desirable. In this study, we demonstrate the potential of pH-responsive biodegradable carbonate apatite (CA) nanoparticles as CpG ODN delivery vehicles that can enhance the production of type-I IFNs (such as IFN-α) relative to that induced by CpG ODNs and can augment the adjuvant effects of CpG ODNs in vivo. In contrast to CpG ODNs, CA nanoparticles containing CpG ODNs (designated CA-CpG) induced significant IFN-α production by mouse dendritic cells and human peripheral blood mononuclear cells in vitro; and production of interleukin-12, and IFN-γ was higher in CA-CpG-treated groups than in CpG ODNs groups. In addition, treatment with CA-CpG resulted in higher cytokine production in draining lymph nodes than did treatment with CpG ODNs in vivo. Furthermore, vaccination with CA-CpG plus an antigen, such as ovalbumin or influenza virus hemagglutinin, resulted in higher antigen-specific antibody responses and CD8+ cytotoxic T lymphocyte responses in vivo, in an interleukin-12- and type-I IFN-dependent manner, than did vaccination with the antigen plus CpG ODNs; in addition, the efficacy of the vaccine against influenza virus was higher with CA-CpG as the adjuvant than with CpG ODNs as the adjuvant. These data show the potential of CA nanoparticles to serve as CpG ODN delivery vehicles that increase the production of cytokines, especially IFN-α, induced by CpG ODNs and thus augment the efficacy of CpG ODNs as adjuvants. We expect that the strategy reported herein will facilitate the design and development of novel adjuvant delivery vehicles for vaccines

Induction of Protective Immunity to Listeria monocytogenes with Dendritic Cells Retrovirally Transduced with a Cytotoxic T Lymphocyte Epitope Minigene

In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8(+) T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens

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

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-γ) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-γ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-γ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible