Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

Antiviral properties from plants of the Mediterranean flora.

Natural products are a successful source in drug discovery, playing a significant role in maintaining human health. We investigated the in vitro cytotoxicity and antiviral activity of extracts from 18 traditionally used Mediterranean plants. Noteworthy antiviral activity was found in the extract obtained from the branches of Daphne gnidium L. against human immunodeficiency virus type-1 (EC50 = 0.08 µg/mL) and coxsackievirus B5 (EC50 = 0.10 µg/mL). Other relevant activities were found against BVDV, YFV, Sb-1, RSV and HSV-1. Interestingly, extracts from Artemisia arborescens L. and Rubus ulmifolius Schott, as well as those from D. gnidium L., showed activities against two different viruses. This extensive antiviral screening allowed us to identify attractive activities, offering opportunities to develop lead compounds with a great pharmaceutical potential.

Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection.

OBJECTIVES: To investigate the correlation between the serum levels of the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and the progression of HIV-1 disease. DESIGN: Retrospective analysis of serial serum samples from HIV-1 seroconverters selected according to clinical outcome. METHODS: Twenty-one patients, derived from a cohort recruited between 1985 and 1996 for a prospective study of the natural history of HIV infection, were analysed. All patients had at least one HIV-1-seronegative sample within 1 year prior to the first seropositive test and were followed longitudinally throughout the course of HIV-1 infection (mean follow-up, 73.5 months). Nine were rapid progressors (RP; patients who developed AIDS within 60 months of antibody seroconversion), seven were slow progressors (SP; patients who developed AIDS after 60 months), and five were long-term asymptomatic (LTA; patients with circulating CD4+ cells higher than 400 x 10(6)/l, no signs of HIV disease, no antiretroviral therapy for more than 96 months). A total of 339 serum samples was studied (mean, 16.1 per patient). The levels of RANTES, MIP-1alpha and MIP-1beta were measured by enzyme-linked immunosorbent assay and correlated with different immunological and clinical parameters. RESULTS: Over the entire follow-up period, the geometric mean of serum RANTES was significantly higher in RP [68.6 ng/ml; 95% confidence interval (CI), 56.9-82.7] than in SP (23.7 ng/ml; 95% CI, 20.0-28.2; P < 0.001) and LTA (19.5 ng/ml; 95% CI, 15.5-24.5; P < 0.001). This difference was already significant during the early clinical stages, when patients had peripheral blood CD4+ cell counts still greater than 400 x 10(6)/l (P < 0.001). By contrast, the mean serum levels of MIP-1alpha and MIP-1beta did not differ significantly between the three study groups. Multivariate analysis using the Cox proportional hazard model demonstrated that the mean serum concentration of RANTES before the development of AIDS was independently associated with the time to AIDS (relative risk, 4.5; 95% CI, 1.1-18.2; P = 0.035). In patients with low versus high mean serum RANTES before the fall of CD4+ cells below 400 x 10(6)/l, the median AIDS-free time was 117.5 and 42.7 months, respectively (P = 0.037). CONCLUSION: These data suggest that an elevation of serum RANTES predicts a rapid progression of the disease since the early stages of HIV-1 infection.

Hepatitis C virus. The importance of viral heterogeneity.

Although recent evidence indicates that the quasispecies nature of HCV constitutes a critical strategy for the virus to survive in the host, the mechanisms of viral persistence remain unknown. Similarly, the correlates of immune protection in a limited proportion of individuals who succeed in clearing HCV are still largely undefined. Understanding the mechanisms of sterilizing immunity is essential for devising preventive measures against HCV and unraveling how the virus eludes such immunity. As in other viral infections, the complex interactions between the virus and the host early in the course of HCV infection probably determine the outcome of the disease (i.e., resolution or persistence). The evidence now accumulated on HCV and other models of viral infection is compatible with the hypothesis that both cellular and humoral components are needed for definitive viral clearance. Nevertheless, detailed studies of the specific cellular and humoral immune responses during the incubation period and the acute phase of hepatitis C, in relation to the viral quasispecies evolution and the clinical outcome, are still lacking both in humans and in the chimpanzee model. Until such studies are performed, most ideas of viral clearance mechanisms remain hypothetical, and the immunologic basis of HCV clearance will continue to be inferred from associations rather than from causal relationships.

The aetiology of acute hepatitis in Zimbabwe.

The cause of acute viral hepatitis in 141 patients admitted to both Infectious Diseases Hospitals in Harare (Zimbabwe) was hepatitis A in 44, hepatitis B in 86 and hepatitis Non-A Non-B in 11. The wide distribution of hepatitis A and B viruses and early exposure to both in Zimbabwe are shown by the high positivity rate for anti-HAV antibody in patients under 10 years old (87.5%) and for anti-HBs antibody in patients over 20 (60%). Among the 86 hepatitis B cases, e and delta systems were also investigated: 66 patients (76.5%) were HBeAg positive, six (7%) anti-HBe positive and 14 (16.5%) negative for both; only one was anti-delta positive. Two cases of fulminant liver failure (both occurring in HBsAg and anti-HBc IgM positive, but delta-markers negative patients) and five cases of hepatoma (only one of whom was negative for all HBV markers) are described.

Immunity elicited by hepatitis C virus.

Hepatitis C virus (HCV), the major causative agent of post-transfusion and community-acquired non-A, non-B (NANB), is a single-stranded RNA virus characterized by a high degree of genetic heterogeneity. HCV is endemic worldwide and is a major cause of chronic liver disease and hepatocellular carcinoma. The development of a broadly reactive vaccine is a high priority for the control of HCV infection. In recent years, however, serious concerns have been raised regarding the degree of protective immunity elicited by HCV in the host. Several observations, both in patients and in the chimpanzee model, have suggested a lack of protective immunity against HCV. Chronic HCV infection develops in more than 80% of patients, suggesting that in most cases the immune response of the host fails to mediate resolution of the infection. Cross-challenge studies demonstrated that convalescent chimpanzees are not protected against re-infection with homologous or heterologous HCV strains. Similar evidence has been obtained in polytransfused beta-thalassemic children, in whom re-infection with HCV was associated with multiple episodes of acute hepatitis. Although most of the evidence thus far accumulated suggests that HCV does not elicit a protective immune response, recent studies have provided experimental evidence, both in vitro and in vivo, that HCV infection induces a neutralizing antibody response in humans. However, such antibodies are isolate-restricted and ineffective against variant HCV strains emerging in vivo. Recently, using recombinant envelope proteins of HCV, a successful vaccination of chimpanzees against challenge with a homologous viral strain was reported. Whether this vaccine can provide protection against challenge with a higher infectious dose of the homologous virus or against challenge with heterologous strains of HCV remains to be established. Overall, the data hitherto accumulated indicate that the genetic heterogeneity of HCV will be a major impediment for the development of a broadly reactive vaccine for the control of HCV infection.

[The presence of nucleotide sequences of the hepatitis B virus in the serum of HBsAg-negative blood donors in Sardinia]. / Presenza di sequenze nucleotidiche del virus dell'epatite B nel siero dei donatori di sangue HBsAg negativi in Sardegna.

The presence of HBV DNA was assessed in the serum samples from 878 HBsAg negative Sardinian blood donors. They were composed of 481 (55%) donors selected because of abnormal serum alanine aminotransferase (ALT) levels during routine testing of their blood donation, and of 397 donors (45%) selected on the basis of normal serum ALT activities. HBV DNA sequences were detected in 37 (7.7%) out of 481 subjects with abnormal ALT and in 2 (0.5%) out of 397 subjects with normal ALT. Anti-HBc was detected in 199 (41%) of the 481 subjects with abnormal ALT and in 81 (20%) out of 397 subjects with normal ALT. Among the 39 subjects positive for serum HBV DNA, 12 (31%) were positive for anti-HBc, while 27 (69%) were negative for all serological HBV markers. These data show in Sardinia, where HBV infection is endemic, there is a high frequency of HBsAg negative HBV DNA positive individuals in whom multiplication of HBV may occur without conventional serological HBV markers, suggesting the possible existence of HBV-like viruses which may be responsible for some of the presumed non-A non-B hepatitis.

[Immunological aspects in anti-HIV seronegative drug addicts. Correlation with the duration of heroin addiction and other viral infections]. / Aspetti immunologici nei tossicodipendenti anti-HIV sieronegativi. Correlazione con la durata di assunzione dell'eroina e con altre infezioni virali.

In order to determine whether the immunological abnormalities described in intravenous drug addicts (IDA), are due to HIV infection, other viral infections or to the abuse of narcotic drugs, we studied the T lymphocyte subsets and serological markers of infection with hepatitis B and delta virus, cytomegalovirus and Epstein Barr virus, in 49 IDA. The immunological and serological features of IDA were compared with the control group, made up of 20 healthy subjects. In intravenous drug abusers we found a significant increase in the number of total lymphocytes (P less than 0.01), T-lymphocytes (P less than 0.05), T-suppressor cells (P less than 0.05), and serum IgG levels (P less than 0.0001) as compared with the control group. The prevalence of serological markers of infection with hepatitis B virus, hepatitis delta virus, cytomegalovirus and Epstein Barr virus was significantly higher in IDA as compared with the controls. In conclusion our study demonstrates that T-lymphocyte subsets in IDA seronegative for HIV infection are characterized by an enhancement of peripheral lymphocyte cells with a normal OKT4/OKT8 ratio.

[The presence of nucleotide sequences of the hepatitis B virus in peripheral lymphocytes of patients with acute hepatitis B]. / Presenza di sequenze nucleotidiche del virus dell'epatite B nei linfociti periferici di pazienti con epatite acuta di tipo B.

Hepatitis B virus (HBV) DNA sequences were assessed in 26 patients with acute type B hepatitis, using dot-blot hybridization technique from peripheral blood mononuclear cells (PBMC), during different phases of the illness. At clinical presentation, 15% of patients showed HBV-DNA sequences in PBMC, while serum HBV-DNA was detected in 58% of patients. During clinical improvement 50% of patients had HBV-DNA in PBMC but only 11.5% were positive for serum HBV-DNA. Twenty-three (88.5%) patients recovered and cleared HBV-DNA from serum and from PBMC; three (11.5%) patients with acute hepatitis progressing to chronicity showed persistently HBV-DNA sequences in serum and in PBMC. In conclusion, our study shows that HBV-DNA sequences may be found in PBMC, transiently in patients with acute hepatitis followed by recovery, persistently in patients with acute hepatitis progressing to chronicity.

The use of molecular assays in the management of viral hepatitis.

Molecular assays are instrumental in the clinical management of viral hepatitis. During the past years, a wide variety of molecular assays have been developed and implemented. This considerably improved the understanding of the natural history and pathogenesis of Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Hepatitis delta virus (HDV) hepatitis, but also caused uncertainties in the selection of the most appropriate assays for clinical requirements. Indeed, a rational choice and application of these assays requires adequate knowledge of the performance of the single test. Moreover, the choice of the most accurate assay for patients' needs and physicians' objectives, needs to be oriented to specific contexts, such as diagnosis, management or treatment. In the past, a hurdle in the routine use of assays for hepatitis viruses nucleic acid quantification was represented by the availability of only "home brew" methods which lacked standardization. Major improvement in addressing the use of molecular assays for viral hepatitis has been derived from recent standardization procedures that allowed a comparison between different tests after results were given as International Units. In addition, it should be reminded that, before getting into the market, molecular assays should be approved by European regulation authorities and validated using internationally recognized standards. A subsequent clinical validation should address the diagnostic accuracy of the assay. These proceedings have the aim of identifying which molecular tests, among those currently available, meet clinical requirements for each specific application.

Treatment of chronic hepatitis D.

Despite recent advances in the treatment of chronic viral hepatitis, therapy of chronic hepatitis D is not yet satisfactory. The only option currently available is interferon-alpha (IFN), whose efficacy is related to the dose and duration of treatment. However, the rate of sustained hepatitis D virus (HDV) clearance after a 1-year course with high doses of standard IFN is low. Better results have recently been reported with pegylated IFN both in IFN-naïve and in previous nonresponders to standard IFN, suggesting the use of pegylated IFN as a first-line therapy in chronic hepatitis D. Nucleoside analogues that inhibit hepatitis B virus (HBV) are ineffective against HDV and combination therapy with lamivudine or ribavirin has not shown significant advantages over monotherapy with either standard or pegylated IFN. Because the ultimate goal of treatment is eradication of both HDV and HBV, in responders IFN therapy should be continued as long as possible until the loss of hepatitis B surface antigen, adjusting the dose to patient tolerance. However, because side-effects are common, continuous monitoring is mandatory. Although the first results obtained with pegylated IFN have been encouraging, the rate of sustained virological response is still low and the rate of relapse high, emphasizing the need for developing novel classes of antivirals specifically interfering with the life cycle of this unique virus.

Predicting response to peginterferon alpha-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B.

OBJECTIVE: In a trial of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 24 week post-treatment biochemical and virological response rates with peginterferon alpha-2a with or without lamivudine were significantly higher than with lamivudine alone. The effect of pre-treatment factors on post-treatment responses was investigated. METHODS: Multivariate analyses were performed using available data from 518 patients treated with peginterferon alpha-2a with or without lamivudine, or with lamivudine alone. A post-treatment response was defined as alanine aminotransferase (ALT) normalisation and hepatitis B virus (HBV) DNA level of <20,000 copies/ml. RESULTS: In logistic regression analyses across all treatment arms, peginterferon alpha-2a (with or without lamivudine) therapy, younger age, female gender, high baseline ALT, low baseline HBV DNA and HBV genotype were identified as significant predictors of combined response at 24 weeks post-treatment. In the peginterferon alpha-2a and lamivudine monotherapy arms, patients with genotypes B or C had a higher chance of response than genotype D infected patients (p<0.001), the latter responding better to the combination than to peginterferon alpha-2a monotherapy (p = 0.015). At 1 year post-treatment, response rates by intention-to-treat analysis were 19.2% for the peginterferon alpha-2a, 19.0% for the combination, and 10.0% for the lamivudine groups, with genotypes B or C associated with a sustained combined response to peginterferon alpha-2a with or without lamivudine therapy. CONCLUSIONS: Baseline ALT and HBV DNA levels, patient age, gender, and infecting HBV genotype significantly influenced combined response at 24 weeks post-treatment, in patients treated with peginterferon alpha-2a and/or lamivudine. At 1 year post-treatment HBV genotype was significantly predictive of efficacy for patients treated with peginterferon alpha-2a with or without lamivudine.

The economics of treating chronic hepatitis C patients with peginterferon alpha-2a (40 kDa) plus ribavirin presenting with persistently normal aminotransferase.

Peginterferon alpha-2a (40 kDa) plus ribavirin is effective at achieving sustained viral response compared with no treatment in patients with chronic hepatitis C (CHC) and persistently normal aminotransferase levels (PNALT). The cost-effectiveness of treating CHC in the setting of PNALT has not been assessed. Disease progression in patients with PNALT was simulated in a Markov model. The rate of fibrosis progression, quality of life and costs for each health state were based on literature estimates. The perspective of the Italian National Health Service was adopted and costs (euro 2003) and benefits were discounted at 3%. Sensitivity analyses were performed on important parameters. The primary analysis compared combination therapy with peginterferon alpha-2a (40 kDa) plus ribavirin to no treatment in a cohort of patients with mean age 45 years, and was based on findings from a multinational, randomized trial in patients with PNALT. In genotype 1 patients, the risk of cirrhosis at 30 years is forecast to fall from 32% with no treatment to 19% with combination therapy, increasing quality-adjusted life years (QALYs) by 0.74 years at an incremental cost per QALY gained of euro 16,831. The 30-year risk of cirrhosis in genotype 2 or 3 is projected to fall to 9% with combination therapy, an increase in QALYs of 1.34 years, at an incremental cost per QALY gained of euro 3,000. Thus treatment of PNALT with peginterferon alpha-2a (40 kDa) plus ribavirin is projected to reduce the incidence of cirrhosis, increase life expectancy and have an acceptable cost-effectiveness ratio from a societal perspective.

Clinical significance of hepatitis C virus genotypes and quasispecies.

Hepatitis C virus (HCV) is a major cause of morbidity and mortality worldwide. The infection becomes chronic in about 85% of infected individuals, in the face of a strong humoral and cellular immune response. One of the most important features of HCV is its high degree of genetic variability, which is due to the inherent low fidelity of the viral replication machinery. As a consequence, HCV circulates in vivo as a population of divergent, albeit closely related, genomes exhibiting a distribution that follows the model referred to as a quasispecies. The genetic variability of HCV is complex and has been classified into four hierarchical strata: genotypes, subgenotypes, isolates, and quasispecies. Over the past few years, an extraordinary interest has been focused on the biologic and clinical implications of the genetic variability of HCV. Although there is consensus that the genotypes may influence the out come of antiviral therapy, their clinical significance in the natural history of the disease, as well as in transmission, infectivity, and pathogenesis of HCV infection, remains elusive. Conversely, evidence has accumulated that the quasispecies nature of HCV provides a large reservoir of biologically different viral variants that may have important clinical implications for viral persistence by immune escape mechanisms, for the generation of antiviral drug resistance, and for the development of an effective vaccine. This article reviews the state of the art on the biologic and clinical implications of the genetic variability of HCV.

Epidemiology and clinical course of delta infection in Britain.

This study demonstrates the presence of serological markers of delta infection in HBsAg positive patients throughout the years 1976-1982. As in other Northern European countries, delta infection in Britain is predominantly found in drug addicts and haemophiliacs. delta Ag was detected transiently in serum in a minority of patients presenting with acute hepatitis. In the others and those presenting with chronic liver disease, anti-delta was the only serological evidence of delta infection. Our findings also confirm that delta infection is associated with a more severe and rapidly progressive liver disease.

Prevention and treatment of hepatitis delta virus infection.

The prevention of HDV infection can readily be achieved by immunisation with the envelope proteins (HBsAg/pre-S) of the helper virus (HBV) on which HDV is dependent. In patients with established chronic HBV infection, superinfection with HDV may theoretically be controlled, but not prevented, by immunisation to the internal components of HDV which may be the target of a cytotoxic T-cell response to HDV infected hepatocytes. Such a response, by analogy to the effects of immunisation to HBcAg, may result in rapid lysis of infected hepatocytes thereby limiting the spread of HDV through the liver. In our preliminary experiments using recombinant HDAg (amino-acids 13-76) we have shown that a humoral immune response to HDV prior to HDV superinfection, does not control HDV infection but may facilitate it. We do not know whether this immunisation protocol successfully induced a cytotoxic T-cell response or whether the region of HDAg included is recognised by cytotoxic T-cells and involved in the immune destruction of HDV infected cells. Further studies of the mechanisms of lysis of HDV infected hepatocytes to determine the role, if any, of cytotoxic T-cells or humoral lytic mechanisms, is needed. Although short courses of alpha interferon inhibit HDV replication, relapses are common and amelioration of the inflammatory liver disease is not always seen. Longer periods of therapy may be effective if they can be continued until clearance of the helper virus (HBV), as well as HDV, has occurred. In three patients treated for greater than one year, clearance of HBsAg has occurred. If these patients remain in this state after stopping therapy, further studies of long term alpha interferon therapy may be fruitful.