Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells

The hepatitis B virus (HBV) transgenic mouse (Tg) 50-4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2-month-old and 5-month-old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5-month-old Tg, but not in the 2-month-old Tg. Furthermore, CD4+ T cells from 2-month-old mice, but not those from 5-month-old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.     

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.     

Enhancing the immunogenicity of exogenous hepatitis B surface antigen-based vaccines for MHC-I-restricted T cells

Vaccination with either exogenous hepatitis B surface antigen (HBsAg) lipoprotein particles without adjuvants, or plasmid DNA encoding secreted small HBsAg stimulate long-lasting, potent antibody responses in H-2d (BALB/c) and C57Bl/6 (H-2b) mice. Vaccination with exogenous HBsAg primes MHC-I restricted cytotoxic T lymphocyte (CTL) responses to HBsAg in H-2d but not H-2b mice, while DNA vaccination primes HBsAg-specific CTL responses in both mouse strains. We defined vaccination strategies that could elicit CTL responses to exogenous HBsAg in 'low responder' C57Bl/6 mice. We found that the bacterial plasmid DNA itself, synthetic oligodeoxynucleotides containing immunostimulating sequences, or recombinant Th1 cytokines (IL12, IFNgamma) efficiently support priming of CTL responses to exogenous HBsAg in 'low responder' H-2b mice, but have only minor effects on CTL priming in 'high responder' H-2d mice in the high dose range tested. These molecularly well defined adjuvants can thus efficiently support priming of anti-viral T cell responses under 'low responder' conditions.     

生物可降解微球作为乙型肝炎基因免疫佐剂的研究

探讨生物可降解微球对基因免疫的增强作用。采用有机溶剂蒸发法制备聚乳酸聚乙醇酸共聚 物（ＰＬＧＡ）微球,构建含有乙型肝炎病毒表面抗原Ｓ基因的ｐＲＣ－ＣＭＶ真核表达载体,用微球与基因 载体共孵育法制备其混合物。肌肉注射免疫Ｂａｌｂ／ｃ小鼠。结果表明：微球注射组的血清抗体滴度达到 ｌ：１６００,其效果与乙型肝炎病毒表面抗原加铝佐剂注射组相近,而裸ＤＮＡ注射组没有反应。说明了 生物可降解微球可显著的提高基因免疫的免疫反应。     

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.     

Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance

A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.     

含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

研究了庚型肝炎病毒E2(HGV E2)基因片段作为DNA疫苗的可行性。将来自于质粒pThioHis-E2编码HGV E2的基因片段(559bp)亚克隆到质粒pCMV-S中,使之和HBsAg基因位于同一阅读框,形成重组质粒pCMV-S-E2。用纯化的质粒pCMV-S-E2 DNA注射到昆明小鼠后腿四头肌中来免疫小鼠,同时用pCMV-S作为对照。间隔14天再加强一次免疫。在加强免疫后的第8天眼眶取血。用E2—GST融合蛋白作为固定化抗原,通过ELISA检测受试小鼠的体液免疫应答。结果表明,用质粒pCMV-S-EDNA免疫的小鼠可以产生很强的体液免疫应答。     

Evaluation of a novel non-penetrating electrode for use in DNA vaccination

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.     

Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.     

Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study

A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system. Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation. Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar. The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection. No decrease in the anti-HBs response was observed 6 months after injection. No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations. These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus.     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

NIRF在体内外可明显抑制乙型肝炎病毒抗原的表达

随着对NIRF（Np95/ICBP-90 like RING finger protein）研究的深入,其功能已涉及细胞癌变进程以及表观遗传学等领域. 近期研究显示,NIRF能与HBc (hepatitis B virus core protein )相互结合,但其对乙型肝炎病毒(HBV)抗原表达的影响尚不明确. 本文通过转染pAAV-HBV1.3质粒和高压水动力法尾静脉注射BALB/C小鼠,建立乙型肝炎病毒的细胞和动物模型,研究NIRF对乙型肝炎病毒抗原表达的影响. ELISA检测细胞上清和小鼠血清中HBsAg、HBeAg的分泌和表达情况,Western 印迹或免疫组化染色技术检测HBcAg. 结果显示,乙型肝炎病毒抗原分泌的细胞以及小动物模型建立成功,并且无论在体内外,NIRF都能对它们的表达起抑制作用,期待能为后续的HBV致病机理以及治疗药物的研究提供支持与帮助.     

Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.     

Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles.

Hepatitis delta virus (HDV) has an envelope composed of large-, middle-, and small-form hepatitis B surface antigens (HBsAgs) provided by the helper hepatitis B virus (HBV). In order to examine the roles of individual HBsAgs in HDV assembly, we constructed plasmids containing each specific HBsAg gene and then cotransfected each plasmid with HDV cDNA into a permissive human hepatoma cell line (HuH-7) to examine the effects on HDV production. Results indicated that the plasmids containing only the HBsAg genes were able to complement HDV cDNA as efficiently as the plasmid containing the complete HBV genome in generating HDV-like particles. Moreover, the small-form HBsAg alone was sufficient for HDV packaging. The particles produced from the cotransfection experiments have density and protein composition characteristics similar to those of naturally occurring HDV. With the electron microscope, they were identified as 36- to 38-nm-diameter particles. It was concluded that only the HBsAgs were able to help in the assembly of HDV-like particles.     

Human immunodeficiency virus type 1 major neutralizing determinant exposed on hepatitis B surface antigen particles is highly immunogenic in primates.

Hepatitis B surface antigen (HBsAg) produced by recombinant DNA technology is now widely and safely used worldwide for hepatitis B vaccination. We used the HBsAg particle as a carrier molecule for presentation of selected human immunodeficiency virus type 1 (HIV-1) determinants to the immune system. Immunization of rhesus monkeys with an HBsAg chimera carrying the HIV-1 envelope major neutralizing determinant allowed us to generate proliferative T-cell responses and, in some cases, neutralizing antibodies and antibody-dependent cellular cytotoxicity. Since there is an overlap between populations at risk for hepatitis B virus and HIV, HBsAg recombinant particles may be relevant carriers for HIV-1 epitopes and could offer a new approach to the development of an AIDS vaccine.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.