Antonella Cerino

Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription–polymerase chain reaction (RT‐PCR) in 341 consecutive anti‐HCV–positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations ≤5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome. (HEPATOLOGY 1995;21:285–290.)

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

Molecular epidemiology of hepatitis C virus infection among intravenous drug users

BACKGROUND/AIMS The clinico-pathological features of hepatitis C virus infection in intravenous drug users are different from those found in other hepatitis C virus-infected patients. Our aim was to test whether specific viral variants circulate within this particular patient population. METHODS We studied the distribution of hepatitis C virus genotypes in 90 drug addicts and 484 controls, according to the method described by Okamoto. RESULTS Hepatitis C virus type 1a and 3a infections were more frequent among intravenous drug users than in 125 age-matched controls (48.8% and 21.1% vs 17.6% and 11.2%), accounting for the majority of infections in intravenous drug users. Analysis of hepatitis C virus genotypes according to age showed that, in the general population, hepatitis C virus types 1a and 3a were more prevalent among patients younger than 40 years of age than in older individuals (17.6% and 11.2% vs 1.4% and 0.6%). CONCLUSIONS These findings suggest that hepatitis C virus types 1a and 3a were recently introduced in Italy, presumably via needle-sharing among intravenous drug users, and from this reservoir they are extending to the general population, particularly among younger subjects.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7+) hepatitis C virus (HCV)‐specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue‐homing phenotype of effector memory cells (CCR7–; semi‐effectors). However, since they promptly differentiated into full‐effectors upon IL‐2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL‐2 production may have a pivotal role in generating the large population of semi‐effector CCR7–/IFN‐γ– CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL‐2 production, supporting the evidence that IL‐2 was capable both of pushing semi‐effector CTL to complete their effector cell program and of restoring the HCVcore‐dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.

Hypervariable region 1 of hepatitis C virus: immunological decoy or biologically relevant domain?

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to escape and persistent infection. Moreover, a binding site for neutralizing antibodies (Ab) has been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection showed extensive serological cross-reactivity for unrelated HVR1 peptides in the majority of the patients. A significant correlation was found between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses providing strong evidence in support of the contention that HCV variant selection is driven by the host immune pressure. Monoclonal Ab (mAb) generated following immunization of mice with peptides derived from natural HVR1 sequences also showed cross-reactivity for several HVR1 sequences attesting to the existence of conserved amino acid motifs among different variants. These findings suggest that it is possible to induce a broadly cross-reactive immune response to HVR1 and that this mechanism can be used to generate protective immunity for a large repertoire of HCV variants.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti‐HCV assays, we studied 59 anti‐HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti‐HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C‐terminal truncated soluble E2 (sE2) protein (a.a. 390–683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti‐HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti‐HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti‐HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region. J Med Virol 51:1–5, 1997.

Monoclonal Antibodies with Broad Specificity for Hepatitis C Virus Hypervariable Region 1 Variants Can Recognize Viral Particles

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants.

Hepatitis C virus infection among institutionalised psychiatric patients: a regression analysis of indicators of risk

BACKGROUND/AIMS Institutionalised psychiatric patients are at increased risk of developing chronic infection with hepatitis B virus (HBV). However, little information is available on transmission and epidemiology of hepatitis C virus (HCV) in this setting. The aim of this study was to identify potential risk factors of acquiring HCV infection in two large psychiatric institutions in northern Italy. METHODS We designed a case-control study using randomly selected controls from the same study database, consisting of a total of 1180 patients, in order to satisfy the principle that both cases and controls should be representative of the same base experience. A multiple regression logistic analysis was used to identify features that could predict exposure to HCV as evidenced by the presence of circulating anti-HCV antibodies. RESULTS Anti-HCV was detected in 79 patients (6.7%). The prevalence of viraemia and the distribution of genotypes were very similar to those found in subjects with chronic HCV infection drawn from the same geographical area. Multivariate analysis indicated that a diagnosis of psychosis and a history of trauma were statistically significant independent risk factors associated with a positive anti-HCV result (OR 2.615, 1.273-5.373 95% CI and OR 2.096, 1.133-3.877 95% CI, respectively). CONCLUSIONS The findings of this large epidemiological study show for the first time that prolonged residence in psychiatric institutions does not entail per se a significant risk of acquiring HCV infection. Since transmission of HCV in this setting appears to occur predominantly via classical parenteral routes, simple prophylactic measures appear to be adequate to prevent infection.

Kinetics and Significance of Serum Hepatitis C Virus Core Antigen in Patients with Acute Hepatitis C

ABSTRACT An immunoassay detecting hepatitis C virus core antigen was evaluated for its ability to predict clinical outcome in a series of patients with acute hepatitis C. In subjects who cleared the virus, core antigen was no longer detectable within 16 weeks of onset, whereas considerable fluctuations were noted among patients progressing to chronic hepatitis, one of whom showed consistently negative values despite the intermittent presence of viral RNA.

Monitoring Response to Antiviral Therapy for Patients with Chronic Hepatitis C Virus Infection by a Core-Antigen Assay

ABSTRACT A recently released immunoassay detecting total serum hepatitis C virus (HCV) core antigen was used to prospectively monitor virological responses to antiviral treatment in patients with chronic HCV infection. Sustained responders cleared core protein from serum within the first month of therapy and maintained stably negative values for the entire duration of follow-up after treatment discontinuation. However, patients who relapsed or failed to respond showed transient negative values and could not be accurately discriminated either because of the intrinsic lower sensitivity of the core-antigen assay than those of molecular assays or because of differentially regulated secretion of immunoreactive core protein from infected hepatocytes.