Development of Specific Antibodies to an ARF Protein in Treated Patients with Chronic HCV Infection

ArticleinDigestive Diseases and Sciences 52(9):2427-32 · October 2007with6 Reads
DOI: 10.1007/s10620-006-9630-2 · Source: PubMed
Abstract
The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence with unknown biological function. In this study we sought to characterize the prevalence of specific anti-F antibodies in patients with chronic HCV infection and to analyze the anti-F antibody profile before, during, and after antiviral treatment in order to gain a better understanding of the role of F protein in HCV pathogenesis. Serum samples were collected from 44 patients with chronic HCV infection and from 19 healthy controls. Consecutive samples from 27 patients taken before, during, and after treatment with antiviral therapy. The F and the core proteins were cloned from the HCV genome. The recombinant proteins were expressed in Escherichia coli and affinity purified. A sensitive and specific enzyme-linked immunosorbent assay was developed to assess the prevalence of anti-F antibodies. Eighty-nine percent of chronic HCV patients had evidence of anti-F antibodies, and 95% of them had anti-core antibodies. No correlation of anti-F antibodies was found with response to treatment, genotype, or seroconversion. We conclude that the F protein elicits specific antibodies in most individuals chronically infected with HCV with no correlation with response to treatment. Our results confirm the expression of F protein during natural HCV infection.
    • "Also, while the -2/+1 frame sequence is more variable than the zero frame coding for the core protein, F/ARFP frame is as conserved as some other HCV regions such as NS2 and F/ARFP-reactive antibodies display cross-reactivity across a wide range of different HCV sequences, suggesting the conservation of antigenic determinant(s) [5], [7], [9][10][11], [18][19][20]. The HCV -2/+1 frame has been implicated in hepatocellular carcinoma, p21 modulation, iron metabolism, immune/injury response, as well as chronic infection although it may not correlate with the outcome of PEGylated IFNα plus ribavirin therapy [11], [20][21][22][23][24][25] . However, the biological function of HCV alternate reading frame still remains unclear. "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.
    Full-text · Article · Jul 2016
    • "Since the first domain seems to be a well conn served region among the different analyzed ARFP sequences (Fig. 5a), it could be a suitable candidate epitope for further immunological investigations such as monoclonal antibody production. Several studies already reported the presence of antiiARFP antibodies and TTcell response in some HCVVinfected patients [9, 10, 18, 22, 28]. Zhang et al. [36] detected the antiiARFP antibodies in mice immunized with a DNA vaccine containing the coree No. 2 2012 E1–E2 fragments of the HCV genome. "
    [Show abstract] [Hide abstract] ABSTRACT: Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.
    Article · Apr 2012
    • "It has previously been reported that HCV-infected patients develop specific humoral and cellular responses against the Core+1 antibodies in HCV patients with HCC ARFP/F/core+1 protein (Bain et al., 2004; Chuang & Allain, 2008; Cohen et al., 2007; Komurian-Pradel et al., 2004; Morice et al., 2009; Pawlotsky et al., 2005; Troesch et al., 2005; Varaklioti et al., 2002; Walewski et al., 2001; Wu et al., 2007). However, the significance of circulating antibodies to the ARFP/F/core+1 protein remained unclear. "
    [Show abstract] [Hide abstract] ABSTRACT: The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.
    Full-text · Article · Feb 2011
    G DalagiorgouG DalagiorgouN VassilakiN VassilakiP FokaP Foka+1more author...[...]
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