乙肝治疗性疫苗的研制与临床应用

尽管乙肝预防性疫苗非常有效,我国仍有约9300万乙肝病毒(HBV)携带者。抗病毒治疗疗效有限,患者长期使用容易产生耐药性。因此,研制乙肝治疗性疫苗可补充甚至替代目前的抗病毒治疗。T细胞免疫对于乙肝病毒的控制和清除至关重要,目前已经设计出蛋白疫苗、表位多肽疫苗以及DNA或病毒载体疫苗通过临床试验检验其疗效。将就研制乙肝治疗性疫苗这一日益活跃的领域加以概述,着重评价疫苗的细胞免疫应答和临床疗效。     

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS³ Poisson detection mass spectrometry. Only one epitope (E7_11–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7_11–20), previously used as a vaccine candidate, was neither detected by MS³ on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.     

Cellular activities during a mixed leucocyte reaction in the teleost sea bass Dicentrarchus labrax

In this investigation a number of "in vitro" activities of sea bass peripheral blood leucocytes (PBL) against allogeneic PBL inactivated by irradiation were studied. Stimulator PBL were cultured with inactivated allogeneic PBL, and direct counting of lymphocytes was done after 2 weeks by immunofluorescence and flow cytometry using mAbs DLT15 and DLIg3 specific for T-cells and B-cells, respectively. In a one-way mixed leucocyte reaction (MLR), results showed an increase of T lymphocytes, whereas B lymphocytes had values similar to those in control PBL. The increase of T-cells in MLR cultures was also confirmed using RT-PCR by analyzing the expression of the T-cell receptor (beta-subunit) mRNA. The addition of 5 microg/ml of cyclosporin A (CsA) to the MLR caused a significant decrease in T-cell proliferation. Leucocytes from MLR cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control PBL, raising the possibility of the presence of cytotoxic-like T lymphocytes. Cellular activation of PBL was confirmed in 2 weeks MLR by measuring antibody-induced intracellular Ca(++) mobilization with Fura-2 AM. This work represents the first direct quantitative determination of an "in vitro" T-cell activity in a teleost species.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

Induction of HLA-unrestricted and HLA-class-II-restricted cytotoxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus

We recently reported that immunization with a recombinant MUC-1 vaccinia virus (rVMUC-1) protected C57BL/6 mice from challenge with DF3/MUC-1-positive syngeneic tumors. To elucidate whether anti-MUC-1 tumor immunity, especially MUC-1-specific cytotoxic T lymphocytes (CTI), can be induced in cancer patients by rVMUC-1, we stimulated the peripheral blood lymphocytes from patients with DF3/MUC-1⁺ or DF3/MUC-1 colon carcinomas using the autologous monocytes infected with rVMUC-1 (rVAMN). The stimulated T lymphocytes from two patients with DF3/MUC-1-positive colorectal carcinomas (rVPY+T and rVPW+T) demonstrated HLA-unrestricted cytotoxicity against MUC-1, whereas those from the patient with DF3/MUC-1-negative colon carcinoma (rVPA-T) did not. The HLA-unrestricted cytotoxicity was demonstrated by the CD8⁺ T cells possibly recognizing an epitope present on the tandem repeats. Adoptive immunotherapy who performed three times with patient PY, at 4-week intervals. The adoptive transfer of the first stimulated lymphocytes, demonstrating a high level of HLA-unrestricted cytotoxicity against MUC-1, resulted in the significant reduction of the liver metastasis of patient PY. However, HLA-unrestricted cytotoxicity against MUC-1 was extremely reduced at the second transfer and finally eliminated at the third, whereas the CD4⁺ T cells demonstrating HLA-class-II-restricted cytotoxicity against MUC-1 predominantly proliferated at the third adoptive immunotherapy treatment. The liver metastasis and the serum levels of tumor markers (carcinoembryonic antigen CA19-9) demonstrated a rapid and marked increment after the second transfer and especially after the third. These results suggest that the HLA-unrestricted cytotoxic CD8⁺ T cells against MUC-1, induced in patients with DF3/MUC-1⁺ colorectal carcinomas using rVMUC-1, correlate with the antitumor activity in vivo. Received: 22 October 1997 / Accepted: 24 April 1998     

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.