Rapid and simple detection of IFN‐neutralizing antibodies in chronic hepatitis C non‐responsive to IFN‐α

Abstract

Different mechanisms have been proposed for the failure of interferon (IFN) therapy in patients with chronic hepatitis C and multiple sclerosis, for example, the presence of IFN-neutralizing antibodies. In this study, a novel assay system based on the IFN-inducible Mx-promoter was used to detect IFN-neutralizing antibodies in sera of patients with chronic hepatitis C. To monitor IFN bioactivity in IFN-treated patients, a real-time RT-PCR for MxA gene expression in PBMCs was established. Using these two methods, patients with chronic hepatitis C virus (HCV) infection receiving IFN therapy and patients with treatment induced HCV clearance were monitored. Importantly, neutralizing anti-IFN antibodies were detected in the sera of 3 of 38 chronically HCVinfected patients who failed to respond to therapy but none in sera of patients who cleared HCV after IFN therapy. Interestingly, the presence of these antibodies correlated with the lack of MxA induction in PBMCs after initiation of IFN-a therapy. Retrospective analysis of one patient's sera revealed that the anti-IFN-a antibodies had already developed after the first of four unsuccessful IFN therapies, suggesting that neutralizing antibodies may have contributed to the failure of previous IFN treatments. In summary, a novel screening assay was established that may be helpful for testing IFN non-responders for the presence of clinically relevant anti-IFN-a antibodies and for selecting alternative IFN preparations not neutralized by these antibodies.

Multiple Anti-Interferon Actions of the Influenza A Virus NS1 Protein

“…A reporter plasmid carrying the firefly luciferase (FF-Luc) gene under the control of the Mx1 promoter was created by insertion of a 2,300-bp EcoRI/SmaI fragment of the mouse Mx1 promoter (33) into pGL3 (Promega, Mannheim, Germany), creating pGL3-Mx1P-Luc (35). We also used reporter plasmids carrying the FF-Luc gene under the control of the IFN-␤ promoter (p125Luc; kindly provided by Takashi Fujita [75]), the T7 RNA polymerase promoter (pTM1-FFLuc; kindly provided by Friedemann Weber [74]), an IRF1 promoter (pIRF-1-Luc; kindly provided by Stephen Goodbourn [36]), and the NF-B-responsive element (pNF-B-Luc; Stratagene, Amsterdam, The Netherlands).…”

“…To monitor the interaction of the different NS1 constructs with Flag-CPSF, HA-tagged NS1 cDNAs in pcDNA3 were expressed using a TNT transcription/translation kit (Promega) in the presence of 35 S-labeled methionine. The cDNA coding for Flag-CPSF was expressed by transfection into 293T cells.…”

“…The cell lysate was mixed with the 35 S-labeled NS1 proteins and subjected to coimmunoprecipitation (IP) using a monoclonal anti-Flag antibody coupled to protein A-Sepharose. The beads were washed three times, and the precipitated proteins were analyzed for the presence of 35 S-labeled NS1 by autoradiography and for Flag-CPSF by Western blot analysis using a Flag-specific rabbit antiserum. The numbers indicate the relative intensities of the protein bands, with the strongest signal defined as 100.…”

Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2

“…EF487534.1) were amplified from White Leghorn chicken genomic DNA by PCR using the oligonucleotides ch-P-IFNb-L and ch-P-IFNb-R or ch-P-Mx-L and ch-P-Mx-R, respectively. The fragments were cloned with SmaI and HindII to replace the mouse Mx promoter in pGL3-Mx1P-FF-Luc (a kind gift of Georg Kochs, Department of Virology, University of Freiburg, Germany) (20), resulting in pGL3-P-chIFN-␤-luc and pGL3-P-chMx-luc.…”

A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner

“…HEK293 cells or 104C1 cells were cotransfected with pGL3-Mx1P-FF-Luc reporter plasmid expressing firefly luciferase under the control of the Mx1 promoter (28,29) cloned into pGL3 (Promega), pGL 4.73 encoding Renilla luciferase reporter construct, and either VP40 or VP40 D184N . At 24 h p.t., cells were washed with PBS and incu-bated further for 24 h alone or with a recombinant IFN hybrid (interferon-␣/␤ B/D hybrid; 500 U/ml), which has been shown to have broadhost-range activity in an antiviral assay (30).…”