上海肝病在线 Shanghai Liver Diseases' Online

从HBS AG阴性的隐原性/不明原因性肝硬化和肝癌谈----治疗性复合HBV系列疫苗

临床上，HBS AG阴性和/或HBS AB阳性的不明原因性肝硬化和肝癌颇为多见，因没有明显的其他致肝损伤因素可循，故临床上多将此类肝硬化和肝癌称作为隐原性肝硬化和肝癌。

然而，随着检测技术的发展（HBV DNA PCR法）和肝穿剌活检测的开展，发现其实HBS AG阴性和/或HBS AB阳性的不明原因性肝硬化和肝癌患者中很大一部分是血清和/或肝脏HBV DNA阳性和肝内HBV E/C抗原。而且由于血清HBE AG向HBE AB转换，HBV由野生型向变异型转化，分泌性感染型向非分泌性整合型转化，免疫抑制型向促炎型转化，故多伴慢性炎症和肝硬化及肝癌。

由于HBV清除有赖于机体免疫产生针对HBC/E区（特别是与TH1位点相关HBC 1AA-100AA肽段）的CD4/TH1免疫反应，故治疗性疫苗应重点针对HBV的C区特别是与TH1位点相关HBC 1AA-100AA肽段的抗原特异性免疫功能产生，也就是说新的治疗性疫苗应包括HBC抗原组成成分，并采用TH1反应诱导剂产生全面的TH1样反应（细胞和体液免疫）。

治疗性复合HBV系列疫苗含有激活HBC特异性CD4/TH1的抗原组分-----HBC AG，故其 既能激发／产生多克隆、多位点（针对HBV S、前S1、前S2、C/E、X、DNA多聚酶等多处、多位点） 、强力和全面（细胞与体液）的免疫功能以清除/控制HBV，又能重建/恢复机体的自然免疫功能防止发病。

关于HBS AG阴性和/或HBS AB阳性的不明原因性肝硬化和肝癌检测发现血清和/或肝脏HBV DNA阳性和肝内HBV E/C抗而表明HBV变异株感染有大量报道，兹列一些英文文摘于下以供参阅。

Gastroenterology 2002 Mar;122(3):614-24

Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen.

Lau GK, Suri D, Liang R, Rigopoulou EI, Thomas MG, Mullerova I, Nanji A, Yuen ST, Williams R, Naoumov NV.

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.

精采华丽篇：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

J Virol 1995 Jun;69(6):3358-68

Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection.

Jung MC, Diepolder HM, Spengler U, Wierenga EA, Zachoval R, Hoffmann RM, Eichenlaub D, Frosner G, Will H, Pape GR.

Institute for Immunology, University of Munich, Germany.

Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti-HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection.

精采篇：HBV清除作用与HBC AG/HBe Ag特异性TH/CD4T细胞激活有密切关系，指示今后的HBV治疗性疫苗应重点注意激活机体的针对HBV的C/E区抗原的免疫作用

Eur J Clin Invest 1987 Dec;17(6):515-21

Monoclonal anti-HBs antibodies radioimmunoassay and serum HBV-DNA hybridization as diagnostic tools of HBV infection: relative prevalence among HBsAg-negative alcoholics, patients with chronic hepatitis or hepatocellular carcinomas and blood donors.

Pol S, Thiers V, Nalpas B, Degos F, Gazengel C, Carnot F, Tiollais P, Wands JR, Berthelot P, Brechot C.

Unite d'Hepatologie--INSERM U-99, Hopital Laennec, Paris, France.

Anti-HBs monoclonal antibodies radioimmunoassay (m-RIA) and HBV-DNA hybridization techniques were used to detect HBs antigen (HBsAg)--associated determinants (evidence of HBV on-going infection) and HBV-DNA sequences (evidence of viral multiplication) in the serum samples of 479 patients who were HBsAg negative by standard solid-phase radioimmunoassay. They included 128 alcoholics, 104 patients with chronic hepatitis, fifty-four with an hepatocellular carcinoma, 100 with coagulation disorders and ninety-three blood donors. The aim of this study was the comparison in these populations of the prevalence of the various HBV markers. m-RIA detected HBsAg-associated determinants in 1% of blood donors, 3% of coagulation disorders, 3.1% of the alcoholics, 21.1% of chronic hepatitis and 16.6% of hepatocellular carcinoma; hybridization identified HBV-DNA sequences in 0.9%, 2.2%, 10.9%, 9.6% and 5.5% of these cases, respectively. The combined prevalence of both markers of an on-going HBV infection (with or without viral multiplication) was 14.16%, 26.9% and 22.2% in the latter groups, respectively, as compared with only 3% in patients with coagulation disorders and 2.1% of blood donors. These results confirm the frequency of HBV or HBV-related virus infection in alcoholics, in chronic hepatitis and hepatocellular carcinomas, despite the absence of HBsAg by standard RIA (or even of any other usual marker); this gives further evidence for variations in the expression of HBV infection. A high and quite similar prevalence of usual serum markers and hybridization results was observed in the alcoholics and in the patients with chronic hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2828076

J Med Virol 1987 Oct;23(2):151-5

Hepatitis B virus (HBV)-DNA-positive hepatocellular carcinoma following hepatitis B virus infection in a child.

Giacchino R, Pontisso P, Navone C, Alberti A, Dini G, Facco F, Ciravegna B.

I Clinica Malattie Infettive, Universita di Genova, Istituto G. Gaslini, Italy.

Integrated hepatitis B virus (HBV) DNA sequences were found in neoplastic liver tissue of a hepatitis B surface antigen (HBsAg)-negative child who had previously suffered from HBsAg-positive chronic active hepatitis and was anti-HBs and anti-hepatitis B core (HBc) positive at the time of tumor development. Reintegration pattern was consistent with the presence of a single integration site of the HBV genome into cellular DNA, and clonal proliferation of such infected cells. A normal liver, tested in the same experiment with the same amount of total DNA, was negative for viral DNA sequences. These findings support the possible oncogenic role of HBV in the development of liver cancer, not only in adults, but also in children, even in patients who are negative for HBsAg at the time of tumor diagnosis.

PMID: 2824681

Hepatology 1990 Sep;12(3 Pt 1):575-81

Identification and characterization of intrahepatic hepatitis B virus DNA in HBsAg-seronegative patients with chronic liver disease and hepatocellular carcinoma in Taiwan.

Lai MY, Chen PJ, Yang PM, Sheu JC, Sung JL, Chen DS.

Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Republic of China.

To clarify the role of hepatitis B virus infection in HBsAg-seronegative patients with chronic liver disease and hepatocellular carcinoma in Taiwan, we examined the hepatitis B virus DNA in liver biopsy tissues of 112 patients by Southern blot analysis. The patients studied included 43 patients with nonalcoholic chronic liver disease, 21 patients with hepatocellular carcinoma and 48 control patients with other hepatobiliary and gastrointestinal diseases. To confirm the specificity of the intrahepatic hepatitis B virus DNA signal and to understand the structure of the integrated viral sequences, molecular cloning and DNA sequencing of an integrated hepatitis B virus DNA were done in one patient. Among 13 patients without serological evidence of previous hepatitis B virus infection, no hepatitis B virus sequences were found in the liver. In other HBsAg-negative patients with evidence of previous hepatitis B virus exposure, a substantial positive rate of intrahepatic hepatitis B virus DNA was found (7%). The intrahepatic hepatitis B virus DNA was all in integrated form. The positive rate among patients with nonalcoholic chronic hepatitis and cirrhosis (2%) was not different from that of the control group with other hepatobiliary and gastrointestinal diseases (4%). However, the positive rate of integrated hepatitis B virus DNA between hepatocellular carcinoma patients and nonhepatocellular carcinoma patients was statistically significant (19% vs. 3%, p less than 0.05). Molecular cloning and sequencing of a 3.0 kb EcoRI fragment of an integrated hepatitis B virus DNA from an anti-HBs-positive patient revealed that it was a partial copy of the hepatitis B virus genome.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2169454

Hepatology 1993 Jan;17(1):20-9

Persistence of hepatitis B and hepatitis C viral genomes in primary liver cancers from HBsAg-negative patients: a study of a low-endemic area.

Paterlini P, Driss F, Nalpas B, Pisi E, Franco D, Berthelot P, Brechot C.

INSERM U.75, CHU Necker, Paris, France.

The role of HBV and HCV in the course of primary liver cancer in patients who are negative for HBsAg has been debated. Using a combination of serological and polymerase chain reaction assays, we investigated the association between HCV and HBV infections and primary liver cancer in 24 HBsAg-negative patients living in France. The presence of HCV RNA and HBV DNA sequences was tested for in serum and in tumorous and nontumorous liver samples. Twelve patients had anti-HCV, and 11 patients had anti-HBs and/or anti-HBc. HCV RNA sequences were found in the serum samples of all anti-HCV-positive patients and none of the patients who were negative. Patients with HCV viremia had HCV RNA genomic sequences and presumed replicative intermediates in both tumorous and nontumorous specimens. Sequence analysis of a hypervariable region in the E2/NS1 gene of HCV showed significant variations between the viral molecules isolated from the nontumorous, tumorous and serum samples. This eliminated the hypothesis of the contamination of the tumor by nontumorous cells and serum particles and assessed that liver tumor cells did contain HCV RNA genomes. Eleven of 22 patients tested had HBV DNA in the serum; 5 patients were anti-HBc positive and anti-HBs positive. Patients with HBV viremia had HBV DNA sequences in both tumorous and nontumorous liver specimens. Selective loss of part of the HBV genome in the tumorous tissue of two of these patients suggested HBV DNA persistence in clonally expanded malignant cells. Only 4 of the 22 patients were negative for both viruses. Our results show that HBsAg-negative hepatocellular cancer in France is associated with chronic HBV or HCV infection and, in some cases, both; these findings are consistent with an etiological role for HBV and HCV in HCC that develops in cirrhotic patients living in areas of low prevalence.

PMID: 8380790

Zhonghua Nei Ke Za Zhi 1993 Mar;32(3):152-4

[Identification of latent HBV infection in primary hepatic carcinoma and liver cirrhosis with polymerase chain reaction] [Article in Chinese]

Yang JM, Liu WW. Department of Gastroenterology, South-west Hospital, Third Military Medical College.

In order to explore latent. HBV infection in primary hepatic carcinoma (PHC) and liver cirrhosis (LC), serum HBV X and S gene were tested with polymerase chain reaction in 40 cases of PHC and LC with both negative HBsAg and HBeAg. The positive rates for X and S gene were much the same. The S gene-positive cases were, however, slightly more than the X gene-positive ones. In the cases of PHC and LC with all the five serum immunologic markers negative HBV DNA was found in 56.3% (9/16) and 45.5% (5/11) of the cases respectively. Of those patients with positive anti-HBs or anti-HBe, HBV DNA was found in 4/8 and 1/4 of the cases respectively. It was shown that in the cases of PHC and LC with all the five markers-negative, about half of them seems to be still related with HBV infection. Some of the PHC and LC cases with positive anti-HBs or anti-HBe may also have low level of viral replication. Detection of HBV DNA with multiple genes is of help to identify more PHC and LC patients with latent HBV infection.

PMID: 8222976

Gastroenterol Jpn 1993 Aug;28(4):547-53

High prevalence of hepatitis B and C viral markers in Japanese patients with hepatocellular carcinoma.

Tanaka N, Chiba T, Matsuzaki Y, Osuga T, Aikawa T, Mitamura K.

Department of Medicine, University of Tsukuba, Japan.

In order to assess the etiologic role of hepatitis C virus (HCV) as well as hepatitis B virus (HBV) in the etiology of HCC, we compared the prevalence of HCV-related antibodies (anti-C100-3, anti-CP9, anti-CP10) and HBV-related markers (HBsAg, anti-HBs, anti-HBc) in sera of patients with liver cirrhosis (LC) with (n = 62) and without (n = 54) hepatocellular carcinoma (HCC). In HBsAg-negative cases, at least one HCV-related marker (including HCV RNA) was detected in 92.3% (48/52) of HCC cases and in all of the 44 LC cases without HCC, with no significant difference between these two groups. In HBsAg-positive cases, the prevalence of either one of these HCV-related markers was 40.0% (4/10) both in patients with and without HCC, and there was no significant difference between these two groups. Moreover, in HBsAg-negative cases and 11 cases of positive HCV-related markers, the prevalence of anti-HBs and/or anti-HBc was significantly higher in LC patients with HCC (85.4%) than those without HCC (43.2%, P < 0.001). These results show a high prevalence of hepatitis B and C viral markers in Japanese patients with HCC and further indicate that previous HBV infection is a risk factor in the occurrence of HCC in HBsAg-negative LC and LC with positive HCV-related markers.

PMID: 7690725

Hepatology 1995 Jul;22(1):25-9

Pathogenic role of hepatitis B virus in hepatitis B surface antigen-negative decompensated cirrhosis.

Chung HT, Lai CL, Lok AS.

Department of Medicine, Queen Mary Hospital, University of Hong Kong.

This study was conducted to determine the rate of detection of serum hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg)-negative decompensated cirrhotic patients who had hepatitis B core and/or surface antibodies (anti-HBc and/or anti-HBs), and to compare the outcome of HBsAg-positive cirrhotic patients who did or did not clear HBsAg during follow-up. Six (5%) of 121 HBsAg-positive cirrhotic patients lost HBsAg after 0.2 to 17.1 years (mean, 9.1 +/- 6.2 yr) of follow-up. The cumulative rates of loss of HBsAg at 1, 5, 10, and 15 years were, respectively, 1.3%, 1.3%, 7.4%, and 44.5%. Compared with the patients who remained HBsAg-positive, those who lost HBsAg had milder disease at presentation and significantly longer survival. Of the patients who lost HBsAg, 83% had improvement in liver function after the loss of HBsAg, and all were alive at the time of writing (0.8 to 5.7 years after loss of HBsAg), whereas 27% of those who remained HBsAg-positive had died and 29% had deterioration in liver function. The rate of detection of serum HBV DNA by polymerase chain reaction (PCR) assay was higher in HBsAg-positive cirrhotic patients who lost HBsAg:67% versus cirrhotic patients who had no previous history of chronic HBV infection; 16% (cryptogenic) and 29% (hepatitis C virus and/or alcohol-induced liver disease). In summary, we found that using PCR, serum HBV DNA can be detected in 28% of HBsAg-negative cirrhotic patients who were studied, but the pathogenic significance of such small amounts of virus is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7601418

Trans R Soc Trop Med Hyg 1998 Sep-Oct;92(5):516-7

High prevalence of hepatitis B viral DNA in cirrhotic patients without surface antigen.

Attallah AM, Hussein M, Tabll A, el-Dosoki E, el-Sadany M, el-Ghawalby N.

Gastroenterology Centre, Mansoura University, Egypt.

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.

PMID: 9861366

Liver 1999 Jun;19(3):177-82

Genetic alterations in the S gene of hepatitis B virus in patients with acute hepatitis B, chronic hepatitis B and hepatitis B liver cirrhosis before and after liver transplantation.

Rodriguez-Frias F, Buti M, Jardi R, Vargas V, Quer J, Cotrina M, Martell M, Esteban R, Guardia J.

Biochemistry Department, Hospital General Universitario Valle Hebron, Barcelona, Spain.

BACKGROUND: Several studies have shown that hepatitis B immunoglobulin (HBIG) imposes a selection pressure on the hepatitis B virus (HBV) S gene, and that the emergence of mutations in this region would make reinfection after orthotopic liver transplantation (OLT) possible. AIMS: This study was undertaken to analyze the presence of HBV S-gene mutations in the different stages of HBV infection and the relationship between HBIG therapy and the emergence of mutations in liver transplant recipients. METHODS: The frequency and location of mutations in the coding region of the HBV S gene were studied by PCR and direct sequencing in 30 patients (7 with acute self-limited hepatitis B, 16 with chronic hepatitis B and 7 recipients of (OLT) for HBV-related end stage liver disease who became reinfected). RESULTS: The average number of amino acid changes was higher in patients with a more advanced stage of disease, 0.57 mutations/100 positions in acute hepatitis B and 1.57 in chronic hepatitis B (1.28 in HBeAg-positive and 1.8 in anti-HBe-positive patients). The average number of substitutions in the transplanted patients was 2.7 before OLT and 3 after OLT. No amino acid substitutions were detected in the "a" determinant of HBsAg in acute hepatitis B, however, 8 substitutions were observed in 6 chronic patients. In 3 OLT patients, 4 substitutions were observed in samples before and after OLT. One of these patients, who had protective levels of anti-HBs, showed 3 additional new amino acid substitutions after OLT, suggesting escape mutant selection by the effect of HBIG therapy. No changes were observed between the consensus sequences obtained several years before and after transplantation, indicating consensus sequence stability. CONCLUSION: These results show that there is an accumulation of HBV S-gene mutations in HBV-related end-stage liver disease. Prophylaxis with HBIG mainly obtained from acute self-limited hepatitis patients who have a highly homogeneous viral population, may be one factor underlying the reinfection after liver transplantation.

PMID: 10395035

J Med Virol 2000 Oct;62(2):151-8

Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53.

Shiota G, Oyama K, Udagawa A, Tanaka K, Nomi T, Kitamura A, Tsutsumi A, Noguchi N, Takano Y, Yashima K, Kishimoto Y, Suou T, Kawasaki H.

Second Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago, Japan. gshiota@grape.med.tottori-u.ac.jp

Hepatitis B virus (HBV) genome was reported to be detected in serum or liver tissues in hepatocellular carcinoma (HCC) patients negative for hepatitis B surface antigen (HBsAg). Hepatitis B x (HBx) and p53 protein were reported to play an important role in HBV-related hepatocarcinogenesis. To clarify latent HBV infection in HBsAg- and anti-hepatitis C virus (anti-HCV)-negative HCC in a Japanese population and involvement of HBx and p53 protein in these patients, we performed the sensitive and specific nested polymerase chain reaction (PCR) and immunohistochemical analysis. Of 1,024 HCC patients we saw between 1974 and 1998, 66 (6.4%) were negative for HBsAg and anti-HCV. Serum DNA was amplified by nested PCR by using specific primers of surface (S), core (C) and X regions in 26 patients negative for HBsAg and anti-HCV. Eighteen (69%) patients were positive for either S, C, or X region and the results of PCR were confirmed by Southern blotting. Of 18 PCR-positive patients, 3 were positive for anti-HBs and 9 were positive for anti-HBc, however, one was negative for any HBV markers. In HBsAg-negative and PCR-positive patients, the positive rates of expression of HBx and p53 were 8/13 (62%) and 7/13 (54%), being comparable to those in HBsAg-positive HCC patients. The results of the present study suggest that high prevalence of HBV infection is observed in HBsAg-negative HCC in a Japanese population and expression of HBx and p53 is consistent with a role, in these patients, for the transforming ability of these proteins. Copyright 2000 Wiley-Liss, Inc.

PMID: 11002243