New perspectives in vaccination and immunotherapies for HBV infections

AbstractThe woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well recognized animal model. The application of this model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use, for lamivudine-CHO-PreS/S vaccine therapy and WHV particles coupled with HBV derived woodchuck PreS/S antibodies (IC complex) studies, as shown that the PreS/S-CHO vaccine is the first human vaccine able to elicit non sterilizing protection in the woodchuck model. The very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, has confirmed that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered

A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

<p>Abstract</p> <p>Background</p> <p>The E1 protein of Hepatitis C Virus (HCV) can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP), and a C-terminal domain (CT) comprising two segments, a pre-anchor and a trans-membrane (TM) region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1.</p> <p>Results</p> <p>Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis.</p> <p>The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA). This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT region of E1, reducing the fusion process <it>in vitro</it>, strongly reduced the amount of cross-recurrence further supporting interaction between this region and FP.</p> <p>Conclusion</p> <p>Our results support a fusion model for HCV in which the FP and the C-terminal region of E1 are juxtaposed and interact in the post-fusion structure. These findings have general implications for viruses, as any visualization of the post-fusion FP-TM complex has been precluded by the impossibility to obtain crystallised viral fusion proteins containing the trans-membrane region. This limitation gives to sequence based modelling efforts a crucial role in the sketching of a molecular interpretation of the fusion process. Moreover, our data also have a more general relevance for cell biology as the mechanism of intracellular fusion showed remarkable similarities with viral fusion</p

Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).</p> <p>Results</p> <p>First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c) cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB) Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis.</p> <p>Conclusion</p> <p>Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful for selection of antiviral targets.</p

F-FDG Uptake Is Predictive of Poor Survival After Surgery for Large-CellNeuroendocrine-Carcinomas of The Lung: A Bicentric Analysis

Introduction: Large cell neuroendocrine carcinoma (LCNEC) represents a relatively rare and poorly studied entity whose management is not clearly established. The aim of this study was to explore the relationship between preoperative 18F-FDG-PET results, pathological features and long-term survival in a large surgical cohort of LCNEC. Methods: From 06/08 to 06/17, the clinical, radiometabolic, pathological and surgical aspects of 121 LCNEC-patients surgically treated in 2 tertiary centers were retrieved. A Cox regression model was used to identify predictors of survival and Kaplan-Meier method to summarize overall survivals. Results: Mean age and male/female ratio were 63.4±8.3 and 3:1, respectively. The main clinical, radiometabolic and surgical characteristics are reported in Tab.1. Most patients were active/former smokers and presented symptoms at diagnosis. 18FDG-PET/Scan was performed in 65 patients (53.7%) with a mean SUVmax of 10.1 (SD±4.6). Higher SUVmax values (SUVmax >10) were detected in tumors with larger size (p=0.004), advanced p-Stages (p=0.019), presenting necrosis (p=0.077) and with positive staining for CD56 (p=0.025) and TTF-1 (0.063). After surgery (R0 in 91% of cases), 52 (43%) patients had pStage-I while about 35% of patients presented with N1-2 disease. Median, 3-yrs and 5-yrs overall survival was 40 months, 52.2% and 44.6%, respectively. At univariate analysis, the survival was significantly influenced by SUVmax values (p=0.009) and by the presence of vascular invasion at pathological examination (p=0.024). Multivariate analysis showed as the FDG-SUVmax was the only independent variable affecting long-term survival (HR:2.86;C.E.: 1.09-7.47;p=0.032). Conclusions: Patients underwent surgical resection for LCNEC of the lung experienced a poor prognosis (5-yrs survival = 44.6% in this study). High-level FDG accumulation (SUVmax >10) correlates with pathological features and results to be independently predictive of poor survival after surgery. This parameter should be taking into account when planning the best strategy of care

A cohort study to evaluate persistence of hepatitis B immunogenicity after administration of hexavalent vaccines

<p>Abstract</p> <p>Background</p> <p>In 2001, two hexavalent vaccines were licensed in Italy (Hexavac^®, Infanrix Hexa^®), and since 2002 were extensively used for primary immunization in the first year of life (at 3, 5, 11/12 months of age). In 2005, the market authorization of Hexavac^®was precautionary suspended by EMEA, because of doubts on long-term protection against hepatitis B virus. The objectives of this study were to evaluate the persistence of antibodies to anti-HBs, in children in the third year of life, and to investigate the response to a booster dose of hepatitis B vaccine.</p> <p>Methods</p> <p>Participant children were enrolled concomitantly with the offering of anti-polio booster dose, in the third year of life. Anti-HBs titers were determined on capillary blood samples. A booster dose of hepatitis B vaccine was administered to children with anti-HBs titers < 10 mIU/ml, with the monovalent precursor product of the previously received hexavalent vaccine. HBsAb titers were tested again one month after the booster.</p> <p>Results</p> <p>Sera from 113 children previously vaccinated with Hexavac^®, and from 124 vaccinated with Infanrix Hexa^®were tested for anti-HBs. Titers were ≥ 10 mIU/ml in 69% and 96% (p < 0,0001) respectively. The proportion of children with titers ≥ 100 mIU/ml did also significantly differ among groups (27% and 78%; p < 0,0001).</p> <p>Post-booster, 93% of children achieved titers ≥ 10 mIU/ml, with no significant difference by vaccine group.</p> <p>Discussion</p> <p>Fifteen months after third dose administration, a significant difference in anti-HBs titers was noted in the two vaccine groups considered. Monovalent hepatitis B vaccine administration in 3-year old children induced a proper booster response, confirming that immunologic memory persists in children with anti-HBs titers < 10 mIU/ml. However, long-term persistence of HBV protection after hexavalent vaccines administration should be further evaluated over time.</p

Modulation of RANTES expression by HCV core protein in liver derived cell lines

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL).</p> <p>Methods</p> <p>HepG2 and Chang Liver (CHL) cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU) of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis.</p> <p>Results</p> <p>Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter.</p> <p>Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates some interferon response genes and acts as a component of cell defense against viral infection.</p> <p>Conclusion</p> <p>HCV core protein have opposite effects on the expression of RANTES in different cell types <it>in vitro</it>, possibly reflecting a similar scenario in different microenvironments <it>in vivo</it>.</p

Viral determinants and host immune responses in the pathogenesis of HBV infection

Hepatitis B virus (HBV) is a virus that infects about 350,000,000 people worldwide with a clinical spectrum of acute hepatitis, the healthy carrier state, cirrhosis and hepatocellular carcinoma (HCC). The outcome of HBV infection is the result of complicated viral-host interactions. As in other infections with non-cythopatic viruses, the immune response is thought to play a crucial role in disease pathogenesis but there is increasing evidence that a variety of viral mechanisms, some depending on the function of virally encoded proteins, have a profound impact on the infected hepatocytes, the liver microenvironment, and host anti-viral responses. Indeed, the virus has evolved multiple mechanisms to ensure its success in infecting a susceptible host. The essential aspects of the life cycle of HBV and the host immune response are reviewed and recent new developments in the molecular virology of HBV, including experimental animal models, in the role of accessory viral proteins in disease pathogenesis and HCC development and in the characterisation of the T cell response in the control of HBV infection, are highlighted. (C) 2002 Wiley-Liss, Inc