We examined hepatitis C virus genotypes in 98 American patients with chronic hepatitis C virus infection by means of two methods; restriction fragment length polymorphism analysis and line probe assay, which is based on type-specific sequence variations in the 5' untranslated region. Type 1 was present in 73 patients (74%), type 2 in 15 (15%), type 3 in 6 (6%) and type 4 in 1 (1%). Line probe assay further subdivided type 1 into 1a (n = 35) and type 1b (n = 37) and type 2 into type 2a (n = 6) and 2b (n = 9). Two patients (2%) had both restriction fragment length polymorphism and line probe assay evidence of dual infection (with types 1 and type 2) while another case had both type 1a and 1b by line probe assay. One patient was untypable by either technique. There was no correlation between infecting genotype and presumed cause, serum indexes of necroinflammatory activity, or age or sex of the patients studied or known duration of infection. Patients with type 2 hepatitis C virus had more severe liver disease histologically (p = 0.0027) compared with other genotypes but, paradoxically, had significantly lower levels of circulating hepatitis C virus RNA (12.1 +/- 12.8 x 10(5) genome equivalents/ml) than other types (36.4 +/- 44.8 x 10(5) genome equivalents/ml, p < 0.001). Response to interferon was less likely to be sustained in patients infected with type 1 than in those infected with other types (7% vs. 40%, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
We have recently described the production of hepatitis C virus-like particles (HCV-LPs) in insect cells that resemble the putative virions. Here we evaluate the humoral and cellular immunogenicity of the virus-like particles with or without viral p7 protein, a small viral polypeptide that resides between the structural and nonstructural regions of the HCV polyprotein and whose function has not been defined. Immunized BALB/c mice developed high titers of anti-E2 antibodies and virus-specific cellular immune responses including cytotoxic T lymphocytes and T helper responses with gamma interferon production. The virus-like particles without p7 generated a higher cellular immune response with a more T H 1 profile than the particles with p7. Immunization of heat-denatured particles resulted in substantially lower humoral and cellular responses, suggesting that the immunogenicity is strongly dependent on particle Hepatitis C virus (HCV) is a positive-stranded RNA virus that belongs to the Flaviviridae family. The HCV genome contains a single open reading frame coding for a polyprotein that is cleaved into structural and nonstructural proteins by hostand virus-specific proteases. The structural proteins consist of core and 2 envelope glycoproteins E1 and E2. Given the global health burden of HCV infection, there is a paramount need to develop a vaccine against HCV. 1 However, a robust infectious tissue culture system for passaging and expanding the virus and for testing neutralizing antibodies is still lacking. Therefore, newer approaches have to be adopted for HCV vaccine development.Because HCV is an enveloped virus, neutralizing determinants likely reside on the surface of the envelope. The envelope protein E2 of HCV contains highly variable sequences within the N-terminal region (HVR1), which are thought to contain neutralizing B-cell epitopes. 2 Immunization of chimpanzees with recombinant E1/E2 proteins can induce hightiter anti-envelope antibodies but only protects animals from a low-dose viral challenge with the homologous strain. 3 Studies in humans and chimpanzees have indicated that failure to generate multispecific cellular immune responses against HCV in the acute phase of infection is associated with chronicity. 4-7 Therefore, an ideal HCV vaccine should be able to induce strong humoral immune responses against the envelope proteins and to prime broad, HCV-specific T-helper and cytotoxic T-cell responses (for review, see Houghton 8 and Lechmann and Liang 9 ).Virus-like particles are attractive as a recombinant protein vaccine, because they mimic closely the properties of native virions. Papillomavirus-and rotavirus-like particles synthesized in insect cells have been shown to generate protective immunity. [10][11][12] These studies showed that virus-like particles can induce not only high-titer neutralizing antibodies but also strong cytotoxic T lymphocyte (CTL) responses in immunized animals. [13][14][15] Our laboratory has recently reported the synthesis of hepatitis C virus-like particles in insect cel...
Cholestatic patients undergoing surgery have increased mortality and demonstrate clinical features suggestive of adrenal insufficiency. To examine whether cholestasis influences the status of the hypothalamic-pituitary-adrenal axis, we evaluated rats with acute cholestasis caused by bile duct resection (BDR) and sham-operated and unoperated controls. Basal unstressed plasma concentrations of AC`TH and corticosterone were similar in BDR and sham-operated and unoperated control rats. However, exposure of BDR rats to saturated ether vapor resulted in significantly less ACTH and corticosterone release in plasma than in the control animals. To understand the mechanism(s) of decreased HPA axis responsiveness to ether stress in cholestasis, we administered corticotropin-releasing factor (CRF) and measured hypothalamic content, mRNA levels and in vitro secretion of CRF and arginine vasopressin (AVP), the two principal secretagogues of ACTH. In BDR animals, ACTH responses to CRF were decreased and hypothalamic content of CRF and CRF mRNA expression in the paraventricular nucleus were decreased by 25 and 37%, respectively. Furthermore, CRF release from hypothalamic explants of BDR rats was 23% less than that ofcontrols. In contrast to CRF, hypothalamic content of AVP was 35% higher, AVP mRNA in the paraventricular nucleus was increased by 6.6-fold, and hypothalamic explant release of AVP was 24% higher in BDR rats than in control animals. Pituitary ACTH contents were similar in BDR and sham resected rats, but higher than unoperated controls. These findings demonstrate that acute cholestasis in the rat is associated with suppression of hypothalamic-pituitaryadrenal axis responsiveness to stress and demonstrate a dissociation between mechanisms of ACTH regulation mediated by
The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.
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