We have developed a novel tuberculosis (TB) vaccine ; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome or-envelope (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CD8 positive CTL activity against TB antigens and improvement of the histopathological tuberculosis lesions, respectively. The Elispot assay showed that HSP65+IL-12 DNA/ HVJ vaccine induced a greater number of IFN-γ producing T cells than BCG in the mouse model. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses (IFN-γ, IL-2, IL-6 production , and lymphocyte proliferation of cynomolgus monkey). The combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.

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  Parenteral vaccines are used commonly against most of infectious diseases. It is noted that these injection type vaccines are meant to induce protective immunity in the systemic compartment but not aimed at use of the benefits of mucosal immunity as a first line of defense against mucosal infectious diseases such as AIDS, SARS and Influenza. In addition, one of major practical obstacles to current vaccination is storage of the vaccine under refrigeration (or cold-chain) in the developing countries. To overcome these concerns, a plant-based vaccine is considered to be an attractive strategy. Currently, we have developed a rice-based oral vaccine that offers significant advantages over available vaccines. In the rice-based vaccine MucoRice, cholera toxin B subunit (CTB) as the vaccine antigen was accumulated in protein bodies as rice seed storage organella. When orally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches (PPs), and inducing toxin-specific serum IgG and mucosal IgA antibodies with neutralizing activity. Further, MuocRice CTB remained stable state and maintained immunogenicity at room temperture for1.5 years and was protected from pepsin digestion in vitro. Taken together, these findings suggest that MucoRice does not require needle/syringe and cold-chain but induces two layers of immunity in both mucosal and systemic compartments, which is the most effective and highly practical global vaccine to combat emerging and re-emerging infectious diseases.

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  Wilm's tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. WT1 antibodies at higher titers and WT1-specific cytotoxic T lymphocytes (CTLs) at higher frequencies were detected in cancer patients than in healthy donors, indicating that WT1 protein was immunogenic. Furthermore, WT1-specific immune responses are considered to be involved with Graft versus Leukemia effect in the context of hematopoietic stem cell transplantation. These findings provided us with the rationale for cancer immunotherapy targeting WT1. Clinical trials of WT1 peptide vaccination for cancer patients were started, and WT1 vaccination-driven immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Further enhancement of efficacy of WT1 peptide vaccine can be expected by co-administration of WT1-specific helper peptide or anti-cancer chemotherapy agent. WT1 peptide vaccination in the setting of MRD (minimal residual disease) may prolong “progression-free survival time”, or decrease “relapse rate”.

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  We identified glypican-3 (GPC3), as a novel oncofetal antigen, overexpressed specifically in hepatocellular carcinoma (HCC) and melanoma in humans by utilizing genome-wide cDNA microarray analyses of HCC tissues and normal fetal and adult tissues. We also found that GPC3 is a novel tumor marker for HCC and melanoma, and that the pre-immunization of BALB/c mice with dendritic cells pulsed with the H-2K^d-restricted mouse GPC3 298-306 (EYILSLEEL) peptide prevented the growth of tumor expressing mouse GPC3. Because of similarities in the binding peptide motifs between H-2K^d and HLA-A24 (A^*2402), the H-2K^d-restricted GPC3 298-306 peptide thus seemed to be useful for the immunotherapy of HLA-A24⁺ patients with HCC and melanoma. We investigated whether the GPC3 298-306 peptide could induce GPC3 reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A24 (A^*2402)⁺ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice (Tgm) to identify the HLA-A2 (A^*0201)-restricted GPC3 epitopes to expand the applications of GPC3 based immunotherapy to the HLA-A2⁺ HCC patients. We found that the GPC3 144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) Tgm without inducing autoimmunity. In 5 out of 8 HLA-A2⁺ GPC3⁺ HCC patients, the GPC3 144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3 298-306 peptide-reactive CTLs were also generated from PBMCs in 4 of 6 HLA-A24⁺ GPC3⁺ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into NOD/SCID mice. We have recently started a phase I clinical trial of GPC3 peptide vaccine-based immunotherapy of patients with advanced HCC.
  We have also succeeded in inhibition of growth of tumors expressing mouse GPC3 by immunization of mice with dendritic cells differentiated in vitro from mouse embryonic stem cells and pulsed with the GPC3 peptides. Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of patients with HCC and melanoma.

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  In Europe and America, the allergen immunotherapy (AIT) for various hay fevers or allergic rhinitis is enforced as causal treatment methods. However, in Japan, the only AIT is subcutaneous immunotherapy (SCIT) for Japanese cedar pollinosis and not so popular due to the long term treatment and the obscure mechanism of action being elucidated etc.
  We are now advancing research of the new vaccine technnologies which aim at the practical applications of ASIT to Japanese cedar pollenosis. The first candidate vaccine is immunoregulatory liposomes which include antigenic polypeptides and chemicals for the induction of immunoregulatory cells such as invariant natural killer T (iNKT) cells, regulatory dendritic cells and regulatory T (Treg) cells. The liposomes encapsulated ovalbumin (OVA) were prepared and injected intraperitoneally into mice primed with alum-adsorbed OVA. After subsequent challenge with OVA alone, IgE and IgG antibody responses were remarkably suppressed for several months. The results suggest that the immunoregulatory liposomes containing antigenic poplypeptides might suppress on-going IgE antibody formations during the pollen-season and induce long-term immune tolerance after the treatment.
  To elucidate the mechanism of action, next, the spleen cells of the mice treated with the immunoregulatory liposomes were analyzed. As a result, the liposomes were taken into B cells such as marginal zone B (MZB) cells in addition to the dendritic cells or the macrophages. It is demonstrated that IL-10 production after the interaction of the liposomes-captured B cells with iNKT cells are involved in the induction of Treg cells.
  Now, as a vaccine of Japanese cedar pollenosis, the immunoregulatory liposomes encapsulating the designed recombinant Cryj 1-Cryj 2 fusion protein is manufactured so that there may be no risk of anaphylaxis. In a mouse model sensitized with natural Cry j1 and Cry j2 antigens, the vaccine showed the suppression of IgE and IgG antibody responses after the challenge with the antigens. Moreover, oral administration of the vaccine also showed the efficacy for the IgE antibody suppression.
  Taken together, it is suggested that our basic technology for immunoregulatory liposomes can be applicable for any allergen-specific immunotherapies.

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  Monoclonal antibody-based therapy targeting interleukine-6 (IL-6) has been established as a treatment for autoimmune diseases, chronic inflammatory diseases, and lymphoproliferative disease. IL-6 is a multifunctional cytokine which plays pathological roles in Castleman's disease, rheumatoid arthritis, juvenile idiopathic arthritis, and Crohn's disease. Many groups have been exploring the therapeutic approach to block the IL-6 signal, and tocilizumab, a humanized monoclonal antibody against human IL-6 receptor, has been the most intensively studied agent for clinical use. A series of clinical trials of tocilizumab have demonstrated the robustness of safety and efficacy of tocilizumab in the treatment of patients with above diseases. In this review, targeting IL-6 actions as an immunotherapy is discussed.

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  Mixed connective tissue disease (MCTD) includes clinical features of systemic lupus erythematosus (SLE), dermatomyositis/polymyositis (DM/PM), and systemic sclerosis (SSc) occurring in conjunction with a high anti-U1-RNP antibody titer. Childhood MCTD rarely manifests the symptoms and signs of DM/PM and SSc, and mostly does those of SLE. Thus, the diagnosis of childhood MCTD is inevitably based on the two major findings, Raynaud's phenomenon and a high titer of anti-U1-RNP antibody. However, in clinical setting there exist patients who have both anti-dsDNA antibody, a SLE disease-marker, and anti-U1-RNP antibody, a MCTD disease-marker, and thus it is hard to differentiate MCTD patients from SLE. Eighty children were enrolled in this study, and divided into 3 groups ; group A, those who are positive for anti-dsDNA antibody/negative for anti-U1-RNP antibody (48 cases, 60.0%), group B : those who are positive for both anti-dsDNA and anti-U1-RNP antibody (22 cases, 27.5%), group C ; those who are negative for anti-dsDNA antibody/positive for anti-U1-RNP antibody (10 cases, 12.5%), and each of the clinical characteristics among these 3 groups was mutually examined. The results indicated that the frequency of hypocomplementemia in group B was close to group A rather than group C, and the frequencies of both hyper-gamma-globulinemia and Raynaud's phenomenon were very close to group C, but not to group A. On the contrary, the findings which seemed to be specific to MCTD, high titers of speckled type anti-nuclear antibody and rheumatoid factor, located at the middle between group A and group C. Thus, children in group B essentially carried characteristic symptoms and signs of both SLE and MCTD, and it will be difficult to differentiate these two diseases at the onset of the disease. Taken together, children with high titers of both anti-dsDNA antibody and anti-U1-RNP antibody as well as clinical symptoms and signs such as hyper-gamma-globulinemia, Raynaud's phenomenon, membranous nephritis, positive speckled type anti-nuclear antibody and rheumatoid factor should be followed and treated as children with MCTD along with SLE.

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  We report a case of classic polyarteritis nodosa complicated with renal infarction. A 14-year-old boy manifested fever, abdominal pain, watery and bloody diarrhea, and weight loss. Laboratory findings indicated anemia, increased levels of C-reactive protein, and erythrocyte sedimentation rate. Lower gastrointestinal endoscopic examination revealed multiple colorectal ulcerations, and histopathological findings were non-specific, suggesting gastrointestinal involvement of Behcet disease. The patient was referred to our hospital, and suspected to have vasculitis syndrome since the abnormal laboratory findings included persistently increased levels of FDP-E/fibrin monomer as well as inflammatory markers, and the extraordinary high excretion of beta 2-microglobulin, which indicated abrupt and massive expression of HLA class I molecule on endothelial cells due to interferon-gammanemia. To examine the site of vasculitis, 3D-CT angiography was applied to demonstrate bilateral renal infarction and renal artery microaneurysms. Together with the clinical, laboratory, and 3D-CT angiographic findings, he was finally diagnosed as having classic polyarteritis nodosa. After 12 month-course of intravenous cyclophosphamide pulses and prednisolone/azatioprine therapy, complete disappearances of inflammatory manifestations, and renal infarction and microaneurysms were documented. The diagnosis of classic polyarteritis nodosa is frequently delayed because both clinical symptoms and signs, and laboratory findings are not disease-specific, but early diagnosis and treatment are necessary to prevent serious organ damage. In addition to the precise estimation of laboratory findings such as inflammatory markers, and FDP-E/D-dimer/fibrin monomer, the newly developed 3D-CT angiography, a less invasive imaging technique, will be helpful to diagnose patients with classic polyarteritis nodosa, and intervene the disease progression with early and active treatment.

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