IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.     

抗个体型抗体对HBV感染的免疫调节作用——用抗个体型抗体诱生抗—HBs

Jerne~([1])指出,机体对抗原的免疫反应可以通过抗体的个体型(Id)和抗个体型抗体(抗-Id)的相互作用而得到调节。这一理论陆续被许多学者的研究结果所证实。     

Production of hepatitis B surface antigen-pulsed dendritic cells from immunosuppressed murine hepatitis B virus carrier: evaluation of immunogenicity of antigen-pulsed dendritic cells in vivo

Vaccines containing hepatitis B surface antigen (HBsAg) induce antibody to HBsAg (anti-HBs) in most normal individuals and protects them from hepatitis B virus (HBV) infection. However, these vaccines are not efficient at inducing anti-HBs in immunosuppressed individuals, especially in immunosuppressed HBV carriers. The aim of this study was to prepare and to assess the efficacy of a dendritic cell (DC)-based vaccine in an immunosuppressed HBV transgenic mouse (HBV-Tg), an animal model of the HBV carrier state. In order to prepare immunosuppressed HBV-Tg, HBV-Tg were injected with FK-506, an immunosuppressive agent, once daily, intraperitoneally for 15 days. Spleen cells of immunosuppressed HBV-Tg expressed very little mRNAs for interleukin-2 and interferon-gamma. DCs were isolated from the spleen of immunosuppressed HBV-Tg and cultured with HBsAg (100 microg) for 48 h to prepare HBsAg-pulsed DCs. Immunosuppressed HBV-Tg expressing HBsAg in the sera were administered with HBsAg-pulsed DCs or unpulsed DCs or HBsAg in adjuvant for different durations. Immunosuppressed HBV-Tg (n = 8) twice administered with HBsAg-pulsed DCs expressed anti-HBs in the sera within 6 weeks of first injection. Seven of eight immunosuppressed HBV-Tg remained positive for anti-HBs in the sera for the next 12 weeks of observation in spite of receiving daily injection of FK-506 for the entire duration. However, immunosuppressed HBV-Tg administered with unpulsed DCs or HBsAg in adjuvant did not express anti-HBs in the sera. The data show that DCs from immunosuppressed HBV-Tg can be loaded with HBsAg to prepare immunogenic HBsAg-pulsed DCs. HBsAg-pulsed DCs induced anti-HBs in immunosuppressed HBV-Tg. This approach may be of use to induce and maintain anti-HBs in immunosuppressed human HBV carriers.     

人工合成免疫刺激DNA联合HBsAg DNA疫苗在HBV转基因鼠的免疫应答研究

目的 :探讨免疫刺激DNA序列联合基因免疫在HBV转基因鼠的免疫应答。方法 :用人工合成硫代修饰的免疫刺激DNA寡核苷酸 (CpGODN)与HBVS区基因真核表达载体 (V HBs)联合免疫HBsAg转基因鼠 ,通过ELISA观察小鼠血清HBsAg及抗 HBs抗体水平 ,并用免疫组化 (SP法 )及病理HE染色观察小鼠肝组织HBsAg表达量的改变及肝组织炎症活动度。结果 :V HBs联合CpGODN组 6只免疫鼠中有 2只血清抗 HBs抗体阳性 ,其平均效价为 (5 6 2 1± 15 16 )mU ml,血清HBsAg浓度在免疫后第 8周时有 2只转阴 ,而单用V HBs组及V 10 12对照组小鼠血清抗 HBs抗体均阴性、HBsAg含量无明显降低。V HBs +CpGODN组肝组织HBsAg的表达量低于V HBs组及对照组 ,并可见大量炎细胞浸润 ,炎症组织活动度积分明显高于V HBs组及对照组。结论 :CpGODN联合V HBs可增强其免疫应答及抗病毒效应。     

GM—CSF促进乙肝病毒基因疫苗诱导的抗体产生

将构建的编码乙肝病毒PreS2+S蛋白的真核表达质粒注射于C57BL/6小鼠胫前肌内,3d后,在同一部位注射rhGM-CSF。基因疫苗接种2周后,血清中可测到抗-HBs,两月后,抗体水平达到高峰,并保持高水平至少2月以上;肌内注射GM-CSF可提高血清抗-HBs水平,提示GM-CSF可作为基因疫苗的佐剂。     

HBsAg基因疫苗诱导小鼠抗体产生的观察

将携带编码乙型肝炎病毒(HBV)表面抗原主要蛋白的S基因的质粒,直接注射小鼠肌肉中,小鼠4周后产生抗-HBs,阳转率83.3%(5/6),加强组阳转率66.7%(2/3),未加强组阳转率为100%(3/3);两组小鼠抗体持续时间可达28周以上;4周以后小鼠体内未检测到HBsAg.     

Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.     

Antigen-antibody complex as therapeutic vaccine for viral hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-gamma. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the titer of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

Antigen-Antibody Complex as Therapeutic Vaccine for Viral Hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-γ. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the liter of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: Implication of its potential use as a therapeutic vaccine

Fusion of specific antigens to extracellular domain of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA4) represents a promising approach to increase the immunogenicity of DNA vaccines. We evaluated this interesting approach for its enhancement on HBV-specific immune responses and its antiviral effects in HBV transgenic mice. A fusion plasmid encoding the extracellular domain of CTLA4 linked with HBsAg was constructed. Mice were immunized by this fusion plasmid. Vaccination with the CTLA4-fused DNA not only induced much higher level of anti-HBs antibody, but also increased HBsAg-specific CD8+ response as well as CTL response in BALB/c mice. Furthermore, both Th1 and Th2 responses were augmented. In HBV transgenic mice, the levels of circulating HBsAg and HBV DNA replication were down-regulated by induction of higher anti-HBs antibody and HBsAg-specific CD8+ response after vaccination with the fusion plasmid. Thus, the CTLA4-fused DNA vaccine led to breakdown of immune tolerance to viral infection in HBV transgenic mice, which might be used as a therapeutic vaccine in HBV infection.     

用微弹轰击法介导的转基因对小鼠进行乙肝病毒遗传免疫研究

用包裹ＤＮＡ的金微弹轰击耳朵，将表达乙型肝炎病毒表面抗原（ＨＢｓＡｇ）基因的重组质粒转入小鼠皮肤细胞，在小鼠血清中检测ＨＢｓＡｇ及抗－ＨＢｓ抗体。实验组８只小鼠中，有３只在基因转移３天后检测到ＨＢｓＡｇ。１４天再轰击１次。１６天后２只鼠中检测到抗－ＨＢｓ抗体。２８天后４只鼠中检测出抗ＨＢｓ抗体。对照组４只小鼠中ＨＢｓＡｇ及抗－ＨＢｓ抗体均未检测出。本实验为今后进一步分析乙肝病毒的遗传免疫奠定了实验基础，提供了实验依据。     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

DNA-mediated immunization of mice with plasmid encoding HBs antigen.

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.     

携带pcDNA3s与EGFPC3质粒的重组减毒沙门氏菌的免疫性与EGFP的表达

目的研究HBsAg DNA重组减毒鼠伤寒沙门氏菌的免疫性与增强型绿色荧光蛋白(EGFP)基因EGFPC3在小鼠体内的表达。方法将质粒pcDNA3s与pEGFPC3分别电转化减毒鼠伤寒沙门氏菌SL8786,构建重组减毒鼠伤寒沙门氏菌SL8786/EGFPC3与SL8786/pcDNA3s。利用时间分辨荧光免疫分析技术(TRFIA)检测小鼠的血清抗体的含量,以及重组菌引起的T细胞增殖和细胞毒性T淋巴细胞(CTL)应答。用荧光显微镜观察EGFPC3表达。结果口服SL8786/p cDNA3s免疫小鼠后,可诱导产生较强的特异性CTL应答。口服免疫小鼠后第11周抗-HBs含量最高。用流式细胞术检测贴壁培养的2只小鼠的脾细胞中SL8786/EGFPC3阳性率分别为19.20%和17.36%,而SL8786/pcDNA3口服的2只小鼠脾细胞中的EGFP阳性率分别仅为1.95%和1.63%。给小鼠口服SL8786/EGFPC33周后,在小鼠肝脏、脾脏、肾脏、胸腺、十二指肠及肌肉等组织中,均有EGFPC3的表达。结论口服SL8786/pcDNA3s在小鼠体内诱导了特异性细胞和体液免疫应答。携带EGFPC3的重组鼠伤寒沙门氏菌SL8786/EGFPC3在小鼠的部分组织中产生绿色荧光表达,该重组菌的构建为减毒沙门氏菌作为活载体基因工程疫苗的研制提供了一个优良模型。     

A final report on safety and immunogenicity of a bivalent aqueous subunit HBV vaccine

The bivalent form of an aqueous formalin-inactivated hepatitis B vaccine was evaluated for safety and immunogenicity in chimpanzees. To evaluate safety five animals were inoculated intravenously with vaccine containing 500 micrograms HBsAg and two animals with 50 micrograms. None of these animals developed hepatitis or any serologic marker indicative of the presence of residual live virus in the vaccine. Twenty-four animals were used to evaluate immunogenicity and protective efficacy. Seven of these immunized animals produced weak or no anti-HBs responses. Two doses of 50 micrograms HBsAg given subcutaneously 1 month apart protected each of four animals that were challenged with 10(3.5) CID50 HBV at 6 and 12 months after immunization and protected three of four animals challenged at 24 months against development of hepatitis or HBsAg. Three of 4 animals in each group immunized with two doses of 20, 10, or 5 micrograms HBsAg were similarly protected when challenged 6 months after immunization. Thirteen of 20 immunized animals that did not develop HBsAg after challenge with HBV developed anamnestic anti-HBs or anti-HBc responses between 2 and 18 months after challenge, indicating minimal replication of challenge virus. The time of onset and frequency of occurrence of these delayed responses was related to the titer of anti-HBs at the time of challenge. False positive Ausab test results were observed in quarantined chimpanzees. These were neither preceded by appearance of HBsAg nor accompanied by development of anti-HBc. In most cases these reactions were due to a reactant having a sedimentation coefficient and an electrophoretic mobility resembling that of IgM. This reactant generally did not appear to confer resistance to challenge with HBV. The humoral immune response was characterized as being entirely of the IgM class 2 weeks after immunization and switched entirely into the IgG class by 10-12 weeks after vaccine administration. At the time of challenge all animals with antibody had anti-HBs of subtype a.     

In VivoInduction of Specific Cytotoxic T Lymphocytes in Mice and Rhesus Macaques Immunized with DNA Vector Encoding an HIV Epitope Fused with Hepatitis B Surface Antigen

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.     

HCV/HBV复合DNA免疫的初步研究

目的 探讨HCV/HBV复合抗原PCX/S免疫原性。方法 将已证实具有良好免疫原性的人工合成的HCV复合多表位抗原基因PCX与HBV的S抗原基因融合于真核表达载体pcDNA3.0,构建真核表达载体pcDNA-PCXS。大量提取质粒并免疫小鼠,用ELISA的方法检测抗-HBs和抗-HCVAb;~3H-TdR渗入法检测免疫小鼠T淋巴细胞增殖;用~(51)Cr释放法检测免疫小鼠特异性CTLs杀伤作用。结果 免疫后均能检测到抗-HBsAb和抗-HCVAb,抗体水平随着时间的推移逐渐升高,但后者OD_(450nm)平均值低于抗-HBsAb的OD_(450nm)。免疫小鼠诱发针对PCX或S抗原的CTL反应和淋巴细胞转化。结论 复合型PCX/S抗原基因可诱发特异性免疫应答,为HCV/HBV双价疫苗的研究提供一定实验基础。     

Prevention and treatment of hepatitis B in patients on hemodialysis and vaccination of hemodialysis health personnel against hepatitis B

The prevalence and incidence of hepatitis B in hemodialysis patients in Croatia have been estimated to 1.3% and 0.03%, respectively. HBV infection in dialysis patients is usually asymptomatic, has a prolonged course, and progresses to chronic HBsAg hepatitis in 50% of cases. Some 15%-40% of HBsAg carriers on dialysis will develop cirrhosis, liver decompensation or hepatocellular carcinoma. Strict adherence to the standard infection prevention measures, continuous monitoring of HBV markers in patients on hemodialysis, patient and personnel immunization and hepatitis B treatment in hemodialyzed patients are mandatory. Each new patient in a dialysis center must be tested for HBV markers irrespective of prior immunization. All patients in the center should be routinely screened every 3-4 months. HBV immunization is mandatory for all patients on dialysis. In patients with uremia the anti-HBs antibody production is decreased (antibodies will develop in 50%-60% of cases after immunization). It is recommended to immunize all patients with progressive kidney disease, preferably in the preterminal stage. Hepatitis B therapy is recommended in all patients with biopsy proven chronic liver disease. Patients should be treated with standard interferon alpha and/or lamivudine, or peginterferon alpha monotherapy. Hepatitis B treatment is most important in kidney and/or liver transplant candidates. HBV immunization is obligatory for all hospital personnel who are in close contact with infected patients and infective materials.     

Development of antibodies to hepatitis B virus surface antigen in bone marrow transplant recipient following treatment with peripheral blood lymphocytes from immunized donors.

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.     

包裹天然骨架CpG ODN和HBsAg的非磷脂脂质体疫苗的免疫效果

非磷脂脂质体NovasomeR○(Np )是由Brij5 2、胆固醇和油酸组成 ,可作为同时传递佐剂和抗原的载体。我们将HBsAg与天然骨架CpGODN (phosphodiesterCpGODN ,pdCpGODN )包裹于Np后免疫BALB/c小鼠 ,检测其免疫效果。结果显示 ,包裹pdCpGODN和HBsAg的Np在小鼠中诱导了很高滴度的抗 HBs抗体产生并诱生了HBsAgS2 8 3 9特异性的CTL ,而铝佐剂组和仅包裹HBsAg的Np组诱生的抗体滴度较低 ,未检测到CTL活力。抗体亚类分析结果表明包裹pdCpGODN和HBsAg的Np诱生的免疫应答类型与pdCpGODN剂量有关 ,较低剂量 (2 4 μgpdCpGODN )诱生的为IgG2a为主的Th1型应答 ,而较高剂量(4 7μgpdCpGODN )诱生的是Th1/Th2混合型应答。铝佐剂和仅包裹HBsAg的Np组诱生的是以IgG1为主的Th2型应答。此外 ,包裹pdCpGODN和HBsAg的Np免疫小鼠脾淋巴细胞在体外HBsAg刺激培养后特异性增殖并分泌高水平的IFN γ。这些结果表明包裹pdCpGODN和HBsAg的Np能增强HBsAg的免疫原性 ,诱生体液 /细胞免疫均衡应答 ,有可能发展为慢性乙肝的治疗性疫苗