The role of cytotoxic T cells and cytokines in the control of hepatitis B virus infection

The aim of this study was to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) infection. Using transgenic mice that replicate HBV at high levels in the liver as recipients of HBV-specific cytotoxic T cells (CTL), we have shown that the antiviral potential of the CTL is primarily mediated by noncytolytic mechanisms that involve the intra-hepatic production of IFN-gamma by these cells. We also showed that, following antigen recognition, HBV-specific CTL recruit antigen non-specific inflammatory cells that contribute to amplify the liver disease initiated by CTL. These results provided insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV infection.     

Viral hepatitis

Three forms of viral hepatitis can be recognized: hepatitis A, hepatitis B, and hepatitis non-A, non-B. Hepatitis A is caused by a picornavirus, is transmitted by the faceal—oral route, does not become chronic, and no chronic virus carriers exist. The virus can be grown in cell cultures, and killed as well as live attenuated virus vaccines are under development. Hepatitis B is caused by an enveloped virus containing a circular, double-stranded form of DNA. The disease is transmitted parenterally through inoculation of blood or blood products containing virus or through close personal contact with a virus-positive person. Hepatitis B becomes chronic in a certain number of cases and can lead to cirrhosis and primary liver cell carcinoma. The blood and certain body secretions of individuals with a persistent or chronic infection may remain infectious for many years. The hepatitis B virus cannot be grown in cell cultures but the entire genome has been sequenced and cloned in bacterial and eukaryotic cells. An inactivated virus vaccine has been prepared from hepatitis B surface antigen present in the plasma of hepatitis B virus carriers and further vaccines are under development. The agents of hepatitis non-A, non-B have not been identified. It is possible to distinguish between a predominantly parenterally transmitted and an orally transmitted form of hepatitis non-A, non-B. The latter is reported to be caused by a picornavirus that does not, however, have any antigenic relationship with hepatitis A virus.     

Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium

A novel vaccine for hepatitis B virus (HBV) was designed by putting a naked DNA vaccine carrying hepatitis B surface antigen (HBsAg) into live-attenuated Salmonella typhimurium. Mucosal immunization by the oral route in mice showed significantly stronger cytotoxic T lymphocyte (CTL) response than recombinant HBsAg vaccination (P < 0.01 at an effector:target ratio of 100:1), while comparable to intramuscular naked DNA immunization at all effector:target ratios. Contrary to previous reports on naked DNA vaccines given intramuscularly, the IgG antibody response induced by the mucosal DNA vaccine is relatively weak when compared to recombinant HBsAg vaccine (P < 0.001 at day 21). These findings are supported by a high interferon-gamma but a low interleukin-4 level detected in the supernatant of splenic cell cultures obtained from mucosally immunized mice. As distinct to recombinant HBsAg vaccine which is effective for protection, oral mucosal DNA vaccine should be considered as a candidate for therapeutic immunization in chronic HBV infection, donor immunization before adoptive transfer of HBV-specific CTL to HBsAg positive bone marrow transplant recipients, and immunization of non-responders to recombinant HBsAg vaccine. This strongly cellular and relatively absent humoral response may make this vaccine a better candidate as a therapeutic vaccine for chronic HBV carriers than naked DNA vaccines, as the humoral response is relatively less important for the clearance of HBV from hepatocytes, but its presence may lead to side effects such as serum sickness and immune complex deposition in chronic HBV carriers.     

A prospective study of acute viral hepatitis with particular reference to hepatitis A.

In order to investigate the relationship of hepatitis A antigen to viral hepatitis, a prospective study was carried out on 97 patients admitted to Fairfield Hospital, Melbourne, with suspected viral hepatitis, and 3 of their family contacts. Evidence of infection with hepatitis A virus was obtained by detecting hepatitis A antigen in stools, and/or antibody to it in sera, by immune electron microscopy. Infection with hepatitis B virus was determined by testing for hepatitis B surface antigen and antibody in serum, by solid phase radioimmunoassay. Sixteen patients were found to have diseases other than viral hepatitis and 2 patients (child contacts) suffered no illness. There was clinical and/or biochemical evidence compatible with viral hepatitis in 82 patients, of whom 35 were confirmed as having hepatitis A and 31 as having hepatitis B infections. In the remaining 16 patients there was no evidence of infection with either hepatitis A or B virus. It is possible that some of these patients may have been infected with viral agents as yet unidentified.     

Impaired hepatitis B vaccine responses during chronic hepatitis C infection: involvement of the PD-1 pathway in regulating CD4(+) T cell responses

Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4⁺ T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4⁺ T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4⁺ T cells was detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4⁺ T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.     

Global control of hepatitis B virus: does treatment-induced antigenic change affect immunization?

Since its widespread introduction, the hepatitis B vaccine has become an essential part of infant immunization programmes globally. The vaccine has been particularly important for countries where the incidence of hepatitis B virus-related hepatocellular carcinoma is high. Effective treatment options for individuals with chronic hepatitis B infection were limited until 1998 when lamivudine, the first nucleoside analogue drug, was introduced. As a single treatment agent, however, lamivudine has a significant drawback: it induces lamivudine-resistant hepatitis B virus strains that may pose a risk to the global hepatitis B immunization programme. Mutations associated with drug treatment can cause changes to the surface antigen protein, the precise part of the virus that the hepatitis B vaccine mimics. However, the emergence of antiviral drug-associated potential vaccine escape mutants (ADAP-VEMs) in treated patients does not necessarily pose a significant, imminent threat to the global hepatitis B immunization programme. Nonetheless, there is already evidence that current treatment regimens have resulted in the selection of stable ADAP-VEMs. Treatment is currently intended to prevent the long-term complications of hepatitis B virus infection, with little consideration given to potential adverse public health impacts. To address individual and public health concerns, trials are urgently needed to find the optimal combination of existing drugs that are effective but do not induce the emergence of ADAP-VEMs. This paper examines the mechanism of antiviral drug-selected changes in the portion of the viral genome that also affects the surface antigen, and explores their potential impact on current hepatitis B immunization programmes.     

乙型肝炎病毒感染者外周血T细胞亚群和穿孔素-颗粒酶变化

目的探讨HBV感染者外周血单个核细胞（PBMC）中T淋巴细胞亚群分布频率及穿孔素和颗粒酶B表达的变化。方法通过细胞表面标记和细胞内细胞因子染色技术．采用流式细胞术分析HBV感染者PBMC中CD4^＋、CD8^＋T细胞的分布以及穿孔素和颗粒酶B的表达。结果与正常对照相比,急性和慢性乙肝患者CD4^＋T细胞百分率无明显改变,CD8^＋T细胞以及穿孔素和颗粒酶B表达均显著增高（前者P〈0．01,后者P〈0．05）。HBV携带者CD4^＋T细胞显著降低（P〈0．05）,CD8^＋T无明显改变。三组CD4^＋／CD8^＋T比例均显著降低（P〈0．05）。结论与HBV携带者不同,急性和慢性乙肝患者CD8可细胞频数及穿孔素和颗粒酶B表达均明显增高,提示细胞毒T细胞的数量及细胞毒颗粒表达与病毒清除和肝损害相关;急性和慢性患者在增高程度上的差异提示慢性乙肝的细胞免疫和细胞毒反应的不完全。     

乙型肝炎病毒感染者外周血T细胞亚群和穿孔素-颗粒酶变化

目的探讨HBV感染者外周血单个核细胞(PBMC)中T淋巴细胞亚群分布频率及穿孔素和颗粒酶B表达的变化。方法通过细胞表面标记和细胞内细胞因子染色技术,采用流式细胞术分析HBV感染者PBMC中CD4+、CD8+T细胞的分布以及穿孔素和颗粒酶B的表达。结果与正常对照相比,急性和慢性乙肝患者CD4+T细胞百分率无明显改变,CD8+T细胞以及穿孔素和颗粒酶B表达均显著增高(前者P<0.01,后者P<0.05)。HBV携带者CD4+T细胞显著降低(P<0.05),CD8+T无明显改变。三组CD4+/CD8+T比例均显著降低(P<0.05)。结论与HBV携带者不同,急性和慢性乙肝患者CD8+T细胞频数及穿孔素和颗粒酶B表达均明显增高,提示细胞毒T细胞的数量及细胞毒颗粒表达与病毒清除和肝损害相关;急性和慢性患者在增高程度上的差异提示慢性乙肝的细胞免疫和细胞毒反应的不完全。     

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein.

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.     

血液透析患者感染乙型和丙型肝炎病毒的调查

采用基因扩增技术，对医院５８例血液透析患者进行医院性感染乙型和丙型肝炎病毒的调查分析。发现乙型肝炎病毒感染者４例，感染率为６．９０％；丙肝病毒感染者１２例，感染率为２０．６９％。随透析次数的增加，丙肝病毒的感染机率明显增加。提示血液透析患者接受疫苗免疫的重要性。由于透析前患者接受过乙型肝炎疫苗的免疫，调查者中有３４．４８％的患者产生了保护性抗体，且乙型肝炎病毒感染率远低于丙型肝炎。指出了血液透析是传播病毒性肝炎的危险因素，并对其防治措施进行了探讨。     

Humoral response to hepatitis B vaccination and its relationship with T CD45RA+ (naïve) and CD45RO+ (memory) subsets in HIV-1-infected subjects

HIV disease leads to defects in cell-mediated immunity, impairing the immune response to new and recall antigens. We studied 55 HIV 1-infected patients who received of recombinant DNA hepatitis B vaccine and 20 controls. The overall hepatitis B virus (HBV) seroconversion rate was 59%. The median CD4+ T cell count among responders was 452 cell/mm(3), higher than non-responders (359 cells/mm(3)). The HIV plasmaviral loads were higher in non-responders. We concluded that total T CD4 cell count, memory T CD4+ cells and lower plasma viral load among HIV-1-infected subjects treated with HAART could be used to predict the immune response to vaccination with hepatitis B vaccine. Thus, considering cost benefits, HVB vaccination should be preferentially provided to HIV-infected patients with T CD4 cells count over 450 cells/mm(3), preferentially whose under HIV replication controlled.     

Characterization of sequence variations in immunodominant regions of the HBV-nucleocapsid protein as a prerequisite for the development of an epitope-based vaccine

BACKGROUND/AIMS: In hepatitis B virus infection, viral elimination is dependent on an efficient antiviral T cell response which is not detectable in chronic hepatitis B. Therefore, new therapeutic concepts focus on T cell activation, such as epitope-based T cell-targeted vaccines. However, with the development of peptide-based vaccines in mind, viral mutations frequently described in hepatitis B within known immunodominant helper epitopes may have an influence on peptide selection. METHODS: Mutant peptides within immunodominant epitopes (aa 1-20, aa 91-105, and aa 143-157) at position 12, 14, 93, 97, 147, 151, 153, and 155 were tested with peripheral blood mononuclear and specific clone cells for their ability to induce proliferation, produce cytokines, induce T cell receptor down-regulation or antagonize wild-type activity of the hepatitis B core antigen-specific CD4+ T cell clones. RESULTS: Five variants could not induce T cell proliferation or cytokine production when the variants were presented alone. Coincubation with wild-type epitopes leads to T cell activation showing that the variants do not act as T cell receptor antagonists for hepatitis B virus-specific CD4+ T cells. In contrast, five other variants and wild-type peptides stimulated CD4+ T cell proliferation and production of Th1 cytokines. CONCLUSIONS: Our data demonstrate that frequently occurring mutations within immunodominant epitopes have rather a nonstimulatory than a strengthening effect and thus should not included in a vaccine.     

Prevention of perinatal hepatitis B transmission in Haimen City,China: Results of a community public health initiative

In regions where hepatitis B virus (HBV) is endemic, perinatal transmission is common. Infected newborns have a 90% chance of developing chronic HBV infection, and 1 in 4 will die prematurely from HBV-related liver disease. In 2010, the Hepatitis B Foundation and the Haimen City CDC launched the Gateway to Care campaign in Haimen City, China to improve awareness, prevention, and control of HBV infection citywide. The campaign included efforts to prevent perinatal HBV transmission by screening all pregnant women for hepatitis B surface antigen (HBsAg), following those who tested positive, and administering immunoprophylaxis to their newborns at birth. Of 5407 pregnant women screened, 185 were confirmed HBsAg-positive and followed until delivery. At age one, 175 babies were available for follow up testing. Of those, 137 tested negative for HBsAg and positive for antibodies to HBsAg, indicating protection. An additional 34 HBsAg-negative babies also tested negative for antibodies to HBsAg or had indeterminate test results, were considered to have had inadequate immune responses to the vaccine, and were given a booster dose. A higher prevalence of nonresponse to HBV vaccine was observed among babies born to hepatitis B e antigen (HBeAg)-positive mothers and mothers with high HBV DNA titers. The remaining 4 babies tested positive for HBsAg and negative for antibodies, indicative of active HBV infection. The mothers of all 4 had viral loads ≥8 × 10⁶ copies/ml in the third trimester. Although inadequate response or nonresponse to HBV vaccine was more common among babies born to HBeAg-positive and/or high viral load mothers, these risk factors did not completely predict nonresponsiveness. All babies born to HBV-infected mothers should be tested upon completion of the vaccine series to ascertain adequate protection. Some babies of HBeAg-positive mothers with high viral load may still become HBV infected despite timely immunoprophylaxis with HBV vaccine and HBIG.     

Hepatitis delta virus infection in Bombay.

From June 1985 to June 1989, sera from 425 cases of acute viral hepatitis were gathered from 2 hospitals in Bombay; 331 sera were positive for hepatitis B surface antigen and immunoglobulin M anti-hepatitis B core antigen, and the donors' disease was diagnosed as hepatitis B. Anti-hepatitis D virus was found in 124 of these sera, and hepatitis D antigen was present in 24 more, conclusively proving the presence of hepatitis delta infection in association with hepatitis B in Bombay. Among the 425 cases of hepatitis, 39 cases of fulminant hepatitis developed, of whom 31 died. Hepatitis B virus (HBV) was the apparent viral infection in 32 of the fulminant cases, and 20 (63%) of them also showed evidence of hepatitis D virus (HDV) infection, suggesting an aggravation of their clinical course due to concurrent HBV and HDV infections.     

The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model

The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.     

Analyse der Immunantwort bei Hepatitis C

The outcome of Hepatitis C virus infection depends on the interaction of the hosts immune system, particularly the virus specific T cell response, with the virus. An early vigorous multispecific TH1 lymphokine dominated and persistant CD4+ and CD8+ T cell response is associated with elimination (control) of the virus and self limited acute hepatitis C, whereas an absent or weak T cell response is associated with viral persistence and chronic course of disease. The weak immune response during chronic hepatitis C is insufficient to eradicate the virus, at best it may exert a certain control but on the other hand contributes to chronic liver damage. The role of the humoral immune response is not clearly defined yet. Understanding the HCV specific T cell response is of great importance since it paves the way for novel therapeutic and prophylactic vaccine approaches for this devastating disease.     

重组乙型肝炎疫苗(CHO细胞)及其应用

通过重组乙型肝炎(乙肝)疫苗[中国仓鼠卵巢细胞(CHO细胞)]种子细胞、生产工艺验证和临床观察资料的比较,阐明重组乙肝疫苗(CHO细胞)的安全性和有效性,并介绍了第三代重组乙肝疫苗(CHO细胞)(Pre-S/S)。对细胞致瘤性、生产工艺病毒灭活能力、脱氧核糖核酸(DNA)残留量、CHO细胞残留蛋白的考察,证实重组乙肝疫苗(CHO细胞)质量完全达到世界卫生组织(WHO)和欧洲药典标准。该疫苗用于新生儿、儿童、成人预防接种和母婴阻断安全、有效。我国研制的重组乙肝疫苗(CHO细胞)安全、有效,为进一步研制新的预防和治疗用乙肝疫苗奠定了基础。     

A non-human hepadnaviral adjuvant for hepatitis C virus-based genetic vaccines

Human hepatitis B virus (HBV) core antigen (HBcAg) can act as an adjuvant in hepatitis C virus (HCV)-based DNA vaccines. Since two billion people are, or have been, in contact with HBV, one may question the use of human HBV sequences as adjuvant. We herein evaluated non-human stork hepatitis B virus core gene-sequences from stork as DNA vaccine adjuvants. Full-length and fragmented stork HBcAg gene-sequences were added to an HCV non-structural (NS) 3/4A gene (NS3/4A-stork-HBcAg). This resulted in an enhanced priming of HCV-specific IFN-γ and IL-2 responses in both wild-type (wt)- and NS3/4A-transgenic (Tg) mice, the latter with dysfunctional NS3/4A-specific T cells. The NS3/4A-stork-HBcAg vaccine primed NS3/4A-specific T cells in hepatitis B e antigen (HBeAg)-Tg mice with dysfunctional T cells to HBcAg and HBeAg. Repeated immunizations boosted expansion of IFN-γ and IL-2-producing NS3/4A-specific T cells in wt- and NS3/4A-Tg mice. Importantly, NS3/4A-stork-HBcAg-DNA induced in vivo long-term functional memory T cell responses, whose maintenance required CD4⁺ T cells. Thus, avian HBcAg gene-sequences from stork can effectively act as a DNA vaccine adjuvant. This technology can most likely be universally expanded to other genetic vaccine antigens, as this completely avoids the use of sequences from a human virus where a pre-existing immunity may interfere with its adjuvant effect.     

Hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) combine CpG oligodeoxynucletides as a novel therapeutic vaccine for chronic hepatitis B infection

Hepatitis B virus infection is a non-cytopathic hepatotropic virus which can lead to chronic liver disease and hepatocellular carcinoma. Traditional therapies fail to provide sustained control of viral replication and liver damage in most patients. As an alternative strategy, immunotherapeutic approaches have shown promising efficacy in the treatment of chronic hepatitis B patients. Here, we investigated the efficacy of a novel therapeutic vaccine formulation consisting of two HBV antigens, HBsAg and HBcAg, and CpG adjuvant. This vaccine formulation elicits forceful humoral responses directed against HBsAg/HBcAg, and promotes a Th1/Th2 balance response against HBsAg and a Th1-biased response against HBcAg in both C57BL/6 and HBV transgenic mice. Vigorous cellular immune response was also detected in HBV transgenic mice, for a significantly higher number of HBs/HBc-specific IFN-γ secreting CD4+ and CD8+ T cells was generated. Moreover, vaccinated mice elicited significantly intense in vivo CTL attack, reduced serum HBsAg level without causing liver damage in HBV transgenic mice. In summary, this study demonstrates a novel therapeutic vaccine with the potential to elicit vigorous humoral and cellular response, overcoming tolerance in HBV transgenic mice.     

Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers

Hepatitis B virus (HBV) is the major pathogen of chronic hepatitis and liver disease worldwide. Despite the availability of effective vaccines against hepatitis B for many years, over 370 million people remain persistently infected with HBV. Viral persistence is thought to be related to poor HBV-specific T-cell responses. Based on clinical data, the development of efficient methods capable of inducing strong T-cell responses is an important and primary step toward the development of immunotherapeutics against chronic HBV infection. We designed a phase I clinical trial in chronic HBV carriers to assess safety, tolerability and immunogenicity of a DNA vaccine expressing HBV small (S) and middle (preS2 +S) envelope proteins. After occurrence of lamivudine breakthrough, 10 HBeAg positive patients with chronic hepatitis B were followed longitudinally before, during and after DNA vaccine therapy. Immunizations were well tolerated and adverse physical events were mild and considered unrelated to the vaccine. Proliferative responses to hepatitis B surface antigen (HBsAg) were detected in two patients after DNA injections. Following three injections of vaccine, interferon (IFN)-gamma-producing T-cells specific for the preS2 or the S antigen were detectable in 50 and 100% of the patients, respectively. Each patient recognized at least one peptide within the envelope domain encoded by the vaccine. Anti-preS2 antibodies and seroconversion to anti-HBe were detected in two patients. This study shows evidences for the safety and immunological efficacy of HBV-DNA vaccination and demonstrates that DNA vaccination can restore or activate T-cell responses in chronic HBV carriers.