Novel vaccines for the treatment of chronic HBV infection based on mycobacterial heat shock protein 70

Immunogenic peptide-based vaccines can raise significant cellular immune responses. Although cytotoxic T lymphocytes (CTL) peptide epitopes are generally poor immunogens, heat shock protein 70 from Mycobacterium tuberculosis (TBhsp70) can overcome this problem since it is a potent adjuvant that links innate and adaptive immune responses. Our goal is to use TBhsp70 as an adjuvant for development of therapeutic vaccines for chronic Hepatitis B virus infection (HBV). To this end, we genetically fused the HBV core 18-27 peptide (HBcAg((18-27))) as a CTL epitope to the C-terminus of TBhsp70 and expressed the resulting protein in methylotropic yeast Pichia pastoris GS115. At the same time, the TBhsp70-HBcAg((18-27)) peptide complex was reconstituted in vitro. We investigated whether TBhsp70-peptide complex and TBhsp70-peptide fusion protein could generate antigen specific CTL responses in vitro. Dendritic cells (DC) from HLA-A2(+) chronic HBV infection and healthy control pulsed with two vaccines were studied phenotypically by FACS analyses and functionally by cytokine release, and HBV-specific CTL response. Our results demonstrate that two vaccines can activate DC of chronic HBV infection and healthy control by upregulation CD40 and CD86, high production of IL-12p70 and TNF-alpha. Furthermore, autologous T cells with DC stimulated by two vaccines can produce IFN-gamma and generate HBV-specific CTL response. However, capacity for CTL response and cytokines production from HBV infections remained inferior to that of healthy controls. Thus, the strategy of utilizing TBhsp70 may provide a novel design for the development of prophylactic and therapeutic vaccines.     

IgM responses to hepatitis-A virus and hepatitis-B core antigen in acute and chronic liver disease in Malawi; possible role of non-A, non-B, hepatitis

Of 33 patients with acute hepatitis in Malawi, 21 had infection by hepatitis-B virus (HBV), five by hepatitis-A virus (HAV) and seven, who had no markers of current HBV or HAV infections, were probably infected by the agent(s) of non-A, non-B, hepatitis. 87 of 88 sera from persons without liver disease contained antibody to HAV and 49 antibody to hepatitis-B surface antigen (anti-HBs) (six were positive for hepatitis-B surface antigen). The diagnosis of recent infection by HAV was made by detecting HAV-specific IGM in single serum samples and, although such tests showed that HAV caused acute hepatitis, its absence in patients with chronic liver disease suggests that, unlike HBV, infection by HAV does not play a role in chronic liver disease in Malawi. Anti-hepatis-B core antigen (anti-HBc)-specific IgM was detected in 19 of 21 patients with acute HBV infection, in three of five HbsAg-positive patients with cirrhosis, but in none of five HbsAg-positive patients with hepatoma.     

乙型肝炎病毒特异性细胞毒T细胞实验研究技术进展

由于乙型肝炎病毒（HBV）感染中抗原特异性细胞毒T细胞（CTL）在病毒清除和肝细胞损害中的重要作用，使其成为研究热点之一。本文对HBV抗原特异性CTL的实验研究技术进展作一综述。涉及的实验方法包括^51Cr释放实验，乳酸脱氢酶释放法，有限稀释法，MHC—I类分子-肽四聚体，酶免疫斑点技术和流式细胞技术等。上述方法分别用于对HBV抗原特异性CTL表达频率，细胞毒功能，以及CTL表达与分泌细胞因子功能的测定。     

Hepatitis B virus (HBV) markers among patients with chronic liver disease in Kuwait

The prevalence of hepatitis B surface antigen (HBsAg), antibody to the hepatitis B core (anti-HBc) and to surface antigen (anti-HBs), was investigated, using sensitive radioimmunoassay (RIA) systems, among patients with different clinical entities of chronic liver disease in Kuwait, and compared to a control blood donor population. 81% of patients and 44% of the controls had at least one HBV marker. 24% of patients, but none of the controls had both HBsAg and a high titre of anti-HBc in the absence of anti-HBs, suggesting a chronic infection. 31% of our patients with hepatosplenic schistosomiasis, 20% with cryptogenic cirrhosis and chronic active liver disease and 60% with hepatocellular carcinoma had these two markers. HBV antigenaemia was significantly more prevalent among male than among female patients and was particularly high among those less than 35 years old. The high prevalence of the various HBV markers among our patients suggests that HBV is a major factor in the development of chronic liver disease in our area. Furthermore, in view of the high prevalence of antigenaemia in patients with hepatosplenic schistosomiasis, HBV infection must play a concomitant role in the development of a more serious form of chronic liver disease among such patients.     

酶联免疫斑点法检测乙型肝炎病毒感染者抗原表位特异性细胞毒性T淋巴细胞

目的 通过观察乙型肝炎病毒(HBV)特异性细胞毒性T细胞(CTL)抗原表位肽刺激外周血单个核细胞(PBMC)产生γ干扰素(IFN-γ)的能力,分析不同类型HBV感染人群特异性CTL免疫应答差异.方法 合成4条人类白细胞抗原(HLA)-A2限制性HBV特异性CTL抗原表位肽[分别为多聚酶P的575-583序列FLLSLGIHL(Tp),HBsAg28-39序列IPQSLDSWVCTSL(Tel),HBsAg183-191序列FLLTRILTI(Te2)和HBcAg18-27序列FLPSDFFPSV(Tc)].流式细胞术鉴定HLA基因表型.用合成的CTL表位肽分别刺激慢性乙型肝炎组(CHB)、慢性乙型重型肝炎组(CSH),既往HBV感染者(N1)组和健康献血员(N2)的PBMC,采用酶联免疫斑点法(ELISPOT)检测分泌IFN-γ的CTL细胞的频率.结果 (1)HLA-A2基因分布频率:44例CHB组为45.5%(20/44),18例CSH组为55.6%(10/18),10例N1组为60%(6/10),10例N2组全部选择以往研究已确定的HLA-A2阳性者.(2)ELISPOT检测结果:①4条HBV特异性抗原多肽反应阳性率在CHB组、CSH组、N1组和N2组分别为50%(10/20)、10%(1/10)、83.3%(5/6)和10%(1/10).N1组反应阳性率高于CSH组(X2=9.000,P=0.008)和N2组(X2=9.000,P=0.008).②各组对Tp、Te1、Te2和Tc四条肽的平均反应强度用斑点形成细胞(SFC)/106PBMC表示,采用非参数秩和检验,N1组对Tp、Te1、Te2和Tc肽的平均反应强度大于CSH组和N2组(分别为77 SFC/106PBMC vs 10 SFC/106PBMC vs 15 SFC/106PBMC,59 SFC/106PBMC vs 0 SFC/106PBMC vs 0 SFC/106PBMC,100 SFC/106PBMC vs 0 SFC/106PBMC vs 22 SFC/106 PBMC和57 SFC/106 PBMC vs 20 SFC/106 PBMC vs 30 SFC/106 PBMC,均P<0.01).结论 各种类型HBV感染者不论病毒清除与否,都可对HBV特异性多肽产生T细胞免疫反应,这种免疫反应以既往感染者最强,慢性乙型肝炎组较弱,慢性乙型重型肝炎组缺乏,提示HBV特异性CTL应答可能是自身免疫机制清除病毒的重要因素.     

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.     

The delivery of HBcAg via Tat-PTD enhances specific immune response and inhibits Hepatitis B virus replication in transgenic mice

Recent studies have indicated that the therapeutic vaccine based on enhancement of HBV-specific cytotoxic T-lymphocyte (CTL) activity may lead to viral clearance in chronically infected individuals. It is demonstrated that protein transduction domains (PTD) from HIV-1-Tat protein is able to enter cells when combined with exogenous antigens and induce specific CTL responses. We have previously testified that the expressed and purified fusion protein containing Tat-PTD47-57 and HBcAg could enter cytoplasm of dendritic cells, and enhance T cells response to generate HBcAg-specific CTLs efficiently in vitro. In the present study, we evaluated HBcAg-specific immune responses of PTD-HBcAg fusion protein in BALB/c mice and antiviral immunity in HBV transgenic mice. The studies showed that PTD-HBcAg not only induced significantly higher antibody responses, but also increased production of cytokine (IFN-γ, IL-2, IL-4 and IL-10) compared to HBcAg alone and PBS. Moreover, PTD-HBcAg fusion protein increased significantly the percentages of IFN-γ+CD8+ T cells and HBcAg-specific (CTL) responses. Also, enhancement of immune response induced by fusion protein reduced HBV DNA and HBsAg levels and decreased the expression of HBsAg in liver tissue of HBV transgenic mice. In conclusion, PTD-HBcAg fusion protein could enhance not only cell immune response but also humoral immune response, and induce robust specific CTL activity and therapeutic effects in HBV transgenic mice.     

A new approach for therapeutic vaccination against chronic HBV infections

There are currently about 257 million people suffering from chronic HBV infection worldwide. In many cases, an insufficient T cell response is causative for establishment of a chronic infection. To ensure a robust cellular immune response and induction of neutralizing antibodies a novel vaccine platform based on modified cell–permeable HBV capsids was utilized. Cell permeability was achieved by fusion of the membrane–permeable TLM-peptide to HBV core monomers, assembling the capsids. Insertion of a Strep-tagIII into the spike tip domain that protrudes from the capsid surface enables flexible loading with antigens that are fused to streptavidin. In this study, HBV surface antigen-derived PreS1PreS2 domain, fused to monomeric streptavidin, served as cargo antigen. Binding between antigen and capsids was characterized by surface plasmon resonance spectroscopy, electron microscopy and density gradient centrifugation. Confocal immunofluorescence microscopy and in vivo imaging of immunized mice demonstrated membrane permeability of cargo-loaded carriers and spread of antigen over the whole organism. Immunization experiments of mice revealed a robust induction of a specific cellular immune response, leading to destruction of HBV-positive cells and induction of HBV-specific neutralizing antibodies. Membrane permeability of these carriers allows needle-free application of antigen-loaded capsids as evidenced by induction of an HBV-specific CTL response and HBV-specific B cell response after oral or transdermal vaccination.These data indicate that cell–permeable antigen carriers, based on HBV capsids and loaded with HBV antigen, have the capacity to induce a cellular and a neutralizing humoral immune response. In addition, cell permeability of the vaccine platform enables antigen transfer across several cell layers, that could allow oral or transdermal immunization.     

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。     

乙型肝炎病毒感染血清学标志与慢性感染自然史研究进展

乙型肝炎（乙肝）病毒（Hepatitis B Virus,HBV）感染的血清学标志主要有乙肝病毒表面抗原、乙肝病毒表面抗体、乙肝病毒e抗原（Hepatitis B e Antigen,HBeAg）、乙肝病毒e抗体和乙肝病毒核心抗体。慢性HBV感染自然史复杂、多样,与初次感染年龄、HBV载量、基因型、突变位点、HBeAg状态、丙氨酸氨基转移酶（Alanine Aminotransferase,ALT）水平、丙型肝炎病毒和／或丁型肝炎病毒合并感染及宿主免疫状态等有关。慢性HBV感染过程可分免疫耐受期、免疫清除期、低或无病毒复制期和病毒恢复活性期。部分HBeAg阴性、ALT高水平和HBV脱氧核糖核酸高滴度（〉10^5拷贝／ml）患者,可出现肝硬化、肝细胞癌和肝功能衰竭等肝脏相关综合征。现有证据表明,合理使用抗HBV药物可减少后遗症发生率和病死率。     

Immune tolerance against HBV can be overcome in HBV transgenic mice by immunization with dendritic cells pulsed by HBVsvp

In chronic Hepatitis B Virus (HBV) infection the function of dendritic cells (DC), T- and B-cells is impaired. DC vaccination is an option to overcome this. DC pulsed in vitro with HBV sub viral particles (HBVsvp) and used to immunize mice can activate HBV directed humoral and cellular immune responses. In the present study we vaccinated HBV transgenic mice as a model for chronic HBV infection and observed humoral and cellular immune responses. In these mice, the lacking immune response against HBV is mainly due to peripheral tolerance. HBVsvp, together with LPS as a co-activating molecule, were used for pulsing and in vitro activation of DC. HBV transgenic mice were injected with pulsed DC two times. Four weeks after DC vaccination humoral and cellular immune responses, viral antigen levels and liver histology were analyzed. DC vaccinated HBV-transgenic mice developed a strong HBV specific antibody and T-cell response after DC vaccination. Neither circulating HBV antigen levels nor viremia, however, were controlled. No liver damage was observed. These results demonstrate that in vitro activation of DC and loading with HBVsvp can overcome tolerance against HBV and reactivate B- and T-cell responses in HBV transgenic mice, but were not sufficient to lead to virus control in these mice. Vaccination using DC, the key players of cellular and humoral immunity, after in vitro reactivation promises to break tolerance against HBV and may help patients with chronic hepatitis B to clear the infection.     

金昌队列人群HBV感染状况对脂肪肝患病的影响

目的探讨金昌队列人群HBV感染对脂肪肝患病的影响,为脂肪肝的防治提供理论依据。方法以金昌队列基线人群为研究对象,开展流行病学调查、实验室检查和腹部B超检查,描述并比较不同HBV感染模式下的脂肪肝患病率异同,采用logistic回归分析不同HBV感染模式对脂肪肝患病的影响。结果金昌队列基线人群共45605人,男性27917人,女性17688人,男女性比例1.6∶1;总人群年龄均值为46.49岁。队列人群常见8种HBV感染模式中以大三阳(HBsAg、HBeAg和HBcAb阳性)、小二阳(HBsAg和HBcAb阳性)和小三阳(HBsAg、HBeAb和HBcAb阳性)3种模式的脂肪肝患病率较低。HBsAg和HBeAg阳性组脂肪肝患病率低于HBsAg和HBeAg阴性组。logistic回归分析结果显示,小二阳(OR=0.61,95%CI:0.39~0.98)和大三阳(OR=0.52,95%CI:0.30~0.89)是脂肪肝患病的保护因素。结论急性HBV感染可降低脂肪肝的患病率,原因可能与活跃的HBV复制干扰机体脂肪代谢有关。     

Epidemiology and long-term consequences of hepatitis delta virus infection in the Yucpa Indians of Venezuela.

To define better the epidemiology and clinical impact of hepatitis delta virus (HDV) infection among hepatitis B virus (HBV) carriers in less developed countries, the authors prospectively studied a cohort of 216 Yucpa Indian HBV carriers in Venezuela. HBV carriers were followed regularly between 1983 and 1988 by physical examination, laboratory testing for liver enzymes and HBV and HDV markers, and epidemiologic history. Among the cohort, 74 (34%) were initially positive for HDV infection, and 35 additional persons became infected during the study. Risk factors for new HDV infection included living in southern Yucpa villages; being young adults (15-19 years) or young children (1-9 years), and living in a household with a person with acute HDV infection. Persons with HDV infection were at high risk of developing chronic liver disease; 56% of HDV-infected persons had moderate-to-severe chronic liver disease at the end of the study compared with none of the HBV carriers without HDV infection. Mortality rates were 6.9% and 8.8% per year, respectively, among initially HDV-positive HBV carriers and those with new HDV infection, because of rapidly progressive chronic liver disease and fulminant hepatitis; mortality was significantly lower in HBV carriers without HDV infection and in non-HBV carriers. HDV superinfection is a devastating disease in HBV carriers in tropical South America. Prevention of HBV infection with hepatitis B vaccine is the best available tool to reduce the impact of this problem.     

Role of activated CD8+ T cells in the initiation and continuation of hepatic damage.

The cause of alcoholic liver disease (ALD) is multifactorial and poorly understood. It is clear that alcohol alone is not responsible for most of the changes associated with ALD and that cofactors are involved in initiation and production of ALD. One cofactor that has received a great deal of attention recently is the concomitant infection with hepatitis C virus (HCV) and alcohol abuse. The interactive effects of HCV and alcohol abuse are still unclear, but apparently they are the result of an inability of the immune system to control the viral infection and exaggerated hepatocyte damage mediated by either the cells of the inflammatory response or factors produced by the inflammatory cells. This review will focus on one aspect of the possible pathogenic effects associated with alcohol abuse and HCV infection: the possible role of the immune system, notably the cytotoxic T lymphocyte (CTL) response. It is clear that the development of a CTL response is critical for the control of HCV infections, and it is also likely that this response is involved in liver damage. In this review, the evidence that shows the importance of the CD8(+) CTL in viral clearance and the role for pathogenesis will be presented. Findings obtained from animal studies that support the suggestion that activated CD8(+) CTLs can induce liver damage will be presented, as will results of recent studies from my laboratory that provide evidence for an effect of alcohol to enhance the liver damage mediated by activated CD8(+) T cells.     

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。     

Clinical course and consequences of hepatitis B infection

Hepatitis B virus (HBV) is a small enveloped virus containing partially double-stranded DNA. The DNA and HBV-specific DNA polymerase are surrounded by the HBV core antigen (HBcAg), which in turn is surrounded by a lipoprotein envelope containing the HBV surface antigen (HBsAg). Serum of HBV-infected patients contains complete virus particles, as well as non-infectious spherical or filamentous HBsAg particles. Acute hepatitis is characterized by the appearance of serum HBV markers, including HBsAg and IgM anti-HBc, which then disappear during convalescence. Persistence of HBsAg for more than 6 months indicates a carrier state. Chronic hepatitis develops in 90% of newborns who become infected, compared with 29-40% of children infected and 5-10% of adults infected. The immune status of the infected person also influences the development of chronic hepatitis. Chronic HBV infection can be diagnosed by serology (identification of HBsAg and HBV DNA), biochemistry (elevated aminotransferase levels) and liver biopsy. The last is important to assess the severity of disease, its stage and prognosis, and to exclude other hepatic diseases. The outcome of chronic HBV infection varies between individuals, with estimated 5-year survivals of 97% for chronic persistent hepatitis, 86% for chronic active hepatitis, and 55% for chronic active hepatitis with cirrhosis. Treatment with interferon alpha is effective in up to 40% of cases, but in view of the very large number of infected people worldwide, vaccination to prevent spread of the disease is a more cost-effective option.     

外周血单个核细胞在乙肝病毒传播中的作用

在急性乙肝和慢性乙肝患者的外周血单个核细胞都有不同程度的乙型肝炎病毒感染。外周血单个核细胞不但可以从外周血摄取乙型肝炎病毒 ,而且乙型肝炎病毒可在其内复制、转录、翻译并能释放病毒颗粒 ,具有一定的感染性。乙型肝炎病毒感染外周血单个核细胞可逃避免疫反应 ,导致病情的隐匿化。当外周血单个核细胞所处的外环境改变时 ,可导致其再次激活 ,引起肝移植术后乙肝复发、乙肝的输血传播及母婴传播     

Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8⁺ T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.     

Exploring hepatitis B: a neglected disease

Chronic hepatitis B viral (HBV) infection can lead to cirrhosis, liver failure, or hepatocellular carcinoma. In the United States, HBV infection is commonly associated with high-risk behaviors such as intravenous drug use or unprotected sex; but it is not as well-known among health care providers that HBV can be transmitted from mother to baby during birth. Worldwide, the majority of cases of chronic HBV infection are in people who contracted the virus during birth. There is a lack of awareness in the United States that immigrants from HBV-endemic countries may be at high risk for chronic HBV. Thus, at-risk individuals may not be screened for HBV. The most recent Centers for Disease Control and Prevention guidelines recommend HBV screening for all people born in Asia, all U.S.-born persons who were unvaccinated as infants and whose parents were born in regions of high HBV endemicity (> or = 8%), and individuals with parenteral risk factors. Screening for HBV starts with HBsAg (hepatitis B surface antigen), HBsAb (antibody to hepatitis B surface antigen), and total anti-HBc (total antibody to hepatitis B core antigen) testing. For those who are HBV-negative (HBsAg-negative) and have no evidence of prior immunity, the three-part HBV vaccination series is recommended.     

Transmission of hepatitis B and hepatitis delta viruses in the households of chronic hepatitis B surface antigen carriers: a regression analysis of indicators of risk

To evaluate whether clinical and laboratory features of a hepatitis B surface antigen (HBsAg) carrier can predict risks of infection, its chronicity, and the development of liver disease among close contacts, the authors studied a cohort of 994 first degree relatives or cohabitants (household contacts) of 226 non-drug-addicted chronic HBsAg carriers (index cases), of whom 77% had liver disease and 26% were superinfected by hepatitis D virus (HDV). A logistic form of regression analysis was used to assess the role of each feature in the index case as predictor of hepatitis B virus (HBV)- and HDV-related outcomes among household contacts. Six models of risk, expressed as odds ratios, were assessed by multivariate step-down analysis, with the following results. 1) Infection with HBV in the household contact was independently predicted by the index case being son, sibling, spouse, female, or HBV-DNA positive. 2) Chronic HBsAg carriage in the adult household contact was associated with female sex of the index case and with being a sibling; among young subjects, household contacts were more likely to be chronic HBsAg carriers when the index case was the mother, a sibling, or an HBV-DNA-positive subject. 3) HBV-DNA positivity in the young contact was more likely when the index case was HBV-DNA positive and when she was the mother. 4) HBV-DNA positivity in the absence of hepatitis B e antigen (HBeAg) in serum in the index case was not related to a similar pattern of infection in HBsAg-positive contacts. 5) Super-infection with HDV of an HBsAg-positive household contact was significantly predicted by female sex of the index case and by anti-HDV positivity. 6) Chronic liver disease in a contact was predicted only by HDV superinfection of the index case. We conclude that horizontal, nonparenteral transmission of HBV among siblings plays a major role in the household of HBsAg carriers from an intermediate endemicity area.