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上海肝病在线 Shanghai Liver Diseases' Online

人工主动HBV疫苗免疫的供体的外周血淋巴细胞转输是一有效的HBV治疗方法----谈治疗性复合HBV系列疫苗的疫苗组方设计

以往认为肝炎、肝硬化和肝癌时，CD4+/CD8+比例下降，而我们发现肝硬化和肝癌有明显免疫抑制时CD4+CD28+下降而CD4+CD28-和CD4+CD25+CD45RO+明显上升，故CD4总数和CD4+/CD8+比例反而上升----参见相关论文，相关其他论文还在整理中）。加上HBV清除与否与免疫感应功能异常有关，即包括单核细胞功能和CD4+淋巴细胞功能异常有关，因此HBV感染中HBV可特异性CD4+亚类的研究及其意义极为重大。

事实上，国外不少研究发现，CD4+，可通过DC--TH1-BLC轴和DC--TH1-CTC轴参与HBV清除，故不少临床与实验研究（参考文献1-8）发现采用人工主动HBV疫苗免疫的供体的外周血淋巴细胞转输方法，成功清除HBV感染，研究发现CD4+参与过继免疫中HBV清除，而且认为是HBC AG特异性CD4+通过CTL和TH1细胞因子效应而参与HBV清除，而HBS特异性CD4+可能HBV特异性BLC激活参与抗HBS抗体产生。但主动HBV疫苗免疫的供体的外周血淋巴细胞转输有特定医疗设施和HLA/MHC配对条件限制，故临床推广极为困难。

采用治疗性复合HBV系列疫苗，通过其所含促进TH1反应药物组分，如牛磺酸等及促TH1反应的细胞因子，通过抑制Th2/3..反应而促进Th1反应，或解释为抑制Tr(调节性)或Ts（抑制性）或Ti(抑制诱导性)细胞功能活性而促进辅助性（Th细胞）功能活性，产生有利于机体抗病毒状态的内环境，与HBV治疗性疫苗抗原组分一起，发挥清除HBV感染和治疗肝损伤的“一箭双雕”的目的.

1）Shouval D, Adler R, Ilan Y. Adoptive transfer of immunity to hepatitis B virus in mice by bone marrow transplantation from immune donors.Hepatology 1993 Jun;17(6):955-9

Recipients of allogeneic bone marrow transplantation are immunosuppressed as a result of their primary disease and by myeloablative therapy. Such patients are dependent on multiple blood products and are at risk for hepatitis B virus infection. Active immunization against hepatitis B in the immediate pre- and post-transplant periods is ineffective, presumably because of decreased T cell-dependent B-cell responses. This study was designed to evaluate, in a mouse model system, the transfer of immunity against hepatitis B to bone marrow transplant recipients through immunization of bone marrow donors against hepatitis B before transplantation. Bone marrow donor BALB/c mice were immunized with a recombinant hepatitis B vaccine. Seroconversion to HBs antibody occurred within 4 wk of primary immunization, and antibody levels in treated donor mice rose above 300 mIU/ml after a single booster injection. Bone marrow recipient mice, conditioned by sublethal irradiation, were injected intravenously with bone marrow cells obtained from syngeneic HBs antibody-positive immune donors. Antibody was detected in 10% of bone marrow recipients within 30 days of transplantation and in 56% 1 mo after a booster injection that led to a secondary rise in HBs antibody. Adoptive transfer of immunity to hepatitis B also occurred after transplantation of T cell-depleted bone marrow cells from hepatitis B-immune donors, albeit at a lower HBs antibody level. These results indicate that immunity to hepatitis B can be transferred in mice by bone marrow transplantation from hepatitis B-immune donors to immunosuppressed recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

采用HBV免疫的供体/小鼠进行BMT细胞过继免疫，可致受体的抗HBS产生增加，且去除T细胞亦有一定作用，说明DC/APC和T细胞均、有益于抗HBS抗体的产生，说明DC--TH1-BLC轴对于抗病毒免疫体液免疫产生很重要

2）Ilan Y, Nagler A, Adler R, Naparstek E, Or R, Slavin S, Brautbar C, Shouval D. Adoptive transfer of immunity to hepatitis B virus after T cell-depleted allogeneic bone marrow transplantation. Hepatology 1993 Aug;18(2):246-52

Recipients of allogeneic bone marrow transplantation are pancytopenic for several weeks and immunosuppressed for many months as a result of myeloablative therapy required to eliminate the basic disease and to prevent allograft rejection. After bone marrow transplantation, these patients remain profoundly immunosuppressed by the chemotherapy and immunotherapy used as prophylaxis against graft-vs.-host disease, treatment of established disease or both. These patients are usually dependent on multiple blood products and are therefore at risk for hepatitis B virus infection, which may run a fulminant course. Active immunization against hepatitis B virus in the immediate pre-bone marrow transplantation and post-bone marrow transplantation periods was found to be ineffective, probably because of the absence of T cell-dependent B-cell responses, which persists for approximately 1 yr after bone marrow transplantation. We studied adoptive transfer of immunity to hepatitis B virus through bone marrow transplantation in two populations of patients. The first group (A) consisted of 12 pairs of BMT donors and recipients, in which all bone marrow donors were positive for antibodies to HBc and HBs as a result of previously acquired hepatitis B virus infection and resolution; all recipients were negative to antibodies to HBc and HBs. The second group (B) consisted of eight pairs of donors and recipients in which all the donors were actively immunized against hepatitis B virus before bone marrow transplantation; all recipients were negative for all hepatitis B virus markers. All bone marrow transplantation recipients were monitored for antibodies to hepatitis B virus antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

3）Ilan Y, Nagler A, Shouval D, Ackerstein A, Or R, Kapelushnik J, Adler R, Slavin S. Development of antibodies to hepatitis B virus surface antigen in bone marrow transplant recipient following treatment with peripheral blood lymphocytes from immunized donors.Clin Exp Immunol 1994 Aug;97(2):299-302

Bone marrow transplantation (BMT) recipients are immunosuppressed and are at risk for contracting severe infections. Recently, adoptive transfer of immunity against hepatitis B virus (HBV) was documented in BMT recipients receiving bone marrow from 'naturally' HBV-infected individuals who recovered spontaneously, or those transplanted with bone marrow cells obtained from actively immunized donors. Furthermore, reconstitution of the immune system in a BMT recipient who was a hepatitis surface antigen (HBsAg)+/HBV DNA+ carrier with HBV immune bone marrow cells led to clearance of the replicating virus, presumably through adoptive cell-mediated immunotherapy. We report three cases of induction of immunity to HBV by selective adoptive transfer by i.v. injection of peripheral blood lymphocytes (PBL) obtained from BMT donors who were actively immunized against HBV after harvesting of bone marrow. All three BMT recipients developed anti-HBs antibodies. In one BMT case in whom antibodies to HBsAg developed following adoptive transfer of immune PBL, a mild booster effect was documented in the BMT recipient upon immunization with a recombinant hepatitis B vaccine. The two remaining patients lost their antibodies to HBsAg in association with relapse of leukaemia. This immune manipulation may open the door to evaluation of adoptive transfer of immunity to HBV through selective transplantation of HBV immune lymphocytes in selected patients such as those with persistent HBV infection, as well as liver transplant recipients who require protection of the graft against HBV re-infection.

HBV感染受体选择性过继已采用HBV疫苗主动免疫的供者/供体的外周血淋巴细胞（CD4+？？？？）便可成功地治疗受体的HBV感染并产生抗HBV抗体，且因免疫不同功能状态而产生抗HBS抗体与否及消失与否，因此对受体的CD4+治疗或过继已采用HBV疫苗主动免疫的供者/供体的外周血淋巴细胞中CD4+可能是一法，而HBV疫苗尤常规疫苗似可作治疗性疫苗用？？？？

4）Shouval D, Ilan Y.Immunization against hepatitis B through adoptive transfer of immunity. Intervirology 1995;38(1-2):41-6

Clearance of hepatitis B virus (HBV) infection requires an effective T-cell-dependent humoral response that is often defective in HBV carriers and in immunosuppressed patients. We have shown in mice and humans that bone marrow (BM)-derived memory cells, capable of producing antibodies to the HBV envelope and nucleocapsid antigens, are transferable from BM donors (BMD) to their recipients. BMD BALB/c mice were immunized with recombinant HBV surface antigen (HBsAg), and BM from anti-HBs-positive donors was transplanted to irradiated recipient mice, who seroconverted to anti-HBs within 30 days of bone marrow transplantation (BMT), and responded to booster vaccination. In a similar manner, 19/26 human BM recipients, who received their HLA-matched BM from BMDs immunized once with HBsAg, seroconverted within several weeks after BMT. Antibodies to observHBsAg were alsoed in 3 recipients of peripheral blood lymphocytes (PBL) obtained from HLA-matched immunized human donors. Finally, clearance of HBsAg and HBV DNA was observed in an HBsAg carrier with leukemia who received BMT from his HLA-matched anti-HBc+/anti-HBs+ brother. These results indicate that adoptive transfer of immunity to HBV may be achieved through immunization of BM or PBL donors against HBV.

采用过继已采用HBV疫苗主动免疫的供者/供体的外周血淋巴细胞或者接受天然HBV免疫的供体进行BMT,均致HBV感染者的HBS AG清除和抗HBS抗体产生

5）Shouval D, Ilan Y.Transplantation of hepatitis B immune lymphocytes as means for adoptive transfer of immunity to hepatitis B virus. Hepatol 1995 Jul;23(1):98-101

采用过继已采用HBV疫苗主动免疫的供者/供体的外周血淋巴细胞作为一免疫有效转移方法

6） Ilan Y, Nagler A, Zeira E, Adler R, Slavin S, Shouval D.Maintenance of immune memory to the hepatitis B envelope protein following adoptive transfer of immunity in bone marrow transplant recipients. Bone Marrow Transplant 2000 Sep;26(6):633-8

Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) has been documented in mice and humans. In the present study, we report long-term follow-up of antibodies to HBsAg in humans who received allogeneic bone marrow transplantation (BMT) from donors immunized with HBsAg. BM donors were immunized with recombinant HBsAg. BM or PB cells were transplanted to HLA matched recipients. Recipients were followed for anti-HBs seroconversion. Control groups included non-immunized or rHBsAg immunized healthy adults as well as individuals that had had hepatitis B and recovered spontaneously. PBLs were stimulated in vitro with rHBsAg and stimulation was expressed as stimulation index. Adoptive transfer of immunity to HBsAg was initially documented in 12 recipients of BM from anti-HBc+/anti-HBs+ donors. An almost 4 year follow-up showed detectable protective anti-HBs levels (>10 mIU/ml) in 50% of patients. Immunity to HBV was also documented in 22/35 BMT recipients (62%), who received their bone marrow from actively immunized donors. In 7/9 of these BMT recipients, anti-HBs antibodies levels were documented 25 months following BMT. In 6/8 (75%) of patients who received only PBLs from HBV immune donors, adoptive transfer of immunity to HBV, and seroconversion to HBsAg+, were documented within 2 months of i.v. injection. Evidence for specific cellular immune response with increased SIs was documented for healthy vaccinees, and BMT recipients, and in none of the healthy non-vaccinated controls. These results suggest that adoptive transfer of immunity to HBV is a useful method for providing long-lasting protection for BM recipients.

采用过继已采用HBV疫苗主动免疫的供者/供体的外周血淋巴细胞或者接受天然HBV免疫的供体进行BMT,均致受体抗HBS抗体产生

7）Bocher WO, Dekel B, Schwerin W, Geissler M, Hoffmann S, Rohwer A, Arditti F, Cooper A, Bernhard H, Berrebi A, Rose-John S, Shaul Y, Galle PR, Lohr HF, Reisner Y. Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection. Eur J Immunol 2001 Jul;31(7):2071-9

Humanized BALB/c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen-loaded dendritic cells together with autologous PBMC from HBV-naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide-specific CTL responses against the immunodominant epitope HBc(18 - 27) were induced by HBc particle or DNA vaccination of chimera engrafted with HBV-naive PBMC. Finally, strong HBc-specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self-limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in our model and clinical studies.

HBc 抗原或 DNA 疫苗接种免疫（DC/APC技术）致TH和抗体及CTL效应产生

8）Lau GK, Suri D, Liang R, Rigopoulou EI, Thomas MG, Mullerova I, Nanji A, Yuen ST, Williams R, Naoumov NV.Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen.Gastroenterology 2002 Mar;122(3):614-24

BACKGROUND & AIMS: Impaired T-cell reactivity is believed to be the dominant cause of chronic hepatitis B virus (HBV) infection. We characterized HBV-specific T-cell responses in chronic hepatitis B surface antigen carriers who received bone marrow from HLA-identical donors with natural immunity to HBV and seroconverted to antibody to hepatitis B surface antigen. METHODS: T-cell reactivity to HBV antigens and peptides was assessed in a proliferation assay, the frequency of HBV core- and surface-specific T cells was quantified directly by ELISPOT assays, and T-cell subsets were analyzed by flow cytometry. RESULTS: CD4+ T-cell reactivity to HBV core was common in bone marrow donors and the corresponding recipients after hepatitis B surface antigen clearance, whereas none reacted to surface, pre-S1, or pre-S2 antigens. Furthermore, CD4+ T cells from donor/recipient pairs recognized similar epitopes on hepatitis B core antigen; using polymerase chain reaction for the Y chromosome, the recipients' CD4+ T lymphocytes were confirmed to be of donor origin. The frequency of core-specific CD4+ and CD8+ T cells was several-fold higher than those specific for surface antigen. CONCLUSIONS: This study provides the first evidence in humans that transfer of hepatitis B core antigen-reactive T cells is associated with resolution of chronic HBV infection. Therapeutic immunization with HBV core gene or protein deserves further investigation in patients with chronic hepatitis B.

接受天然HBV免疫的供体进行BMT致HBV感染者的HBS AG清除，其HBV清除作用与HBC AG特异性TH/CD4T细胞从天然HBV免疫的供体过继给HBV感染的受体有密切关系，即天然HBV免疫的供体的HBC AG特异性TH/CD4T细胞在受体内长期存在于受者体内并发挥其抗HBV免疫的免疫调控作用