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Process Infectivity at Multiple Steps of the Entry Responses and Directly Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral This information is current as Infectivity at Multiple Steps of the Entry of September 26, 2021. Process Chien-Wen S. Kuo, Antonis Mirsaliotis and David W. Brighty Downloaded from J Immunol published online 6 June 2011 http://www.jimmunol.org/content/early/2011/06/06/jimmun ol.1100070 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/06/06/jimmunol.110007 Material 0.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 6, 2011, doi:10.4049/jimmunol.1100070 The Journal of Immunology Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral Infectivity at Multiple Steps of the Entry Process Chien-Wen S. Kuo, Antonis Mirsaliotis, and David W. Brighty Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we Downloaded from have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1–infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is http://www.jimmunol.org/ considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1–infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design. The Journal of Immunology, 2011, 187: 000–000. uman T cell leukemia virus type 1 (HTLV-1) is a del- ease (3, 4). Nonetheless, cytotoxic T cell (CTL) (5–8) and taretrovirus that in some infected individuals causes HTLV-1–specific B cell expansion (9–14) contribute to antiviral H an aggressive adult T cell leukemia/lymphoma or a activity, restrict viral dissemination, and help determine proviral by guest on September 26, 2021 progressive demyelinating disease known as HTLV-1–associated load. Neutralizing Abs are often observed in HTLV-1 infections myelopathy/tropical spastic paraparesis (1, 2). HTLV-1 infections (10–12, 14), and accumulating evidence suggests that a primed are widespread. The virus is prevalent in the Kyushu prefecture of immune response can be protective or prevent infection post- Japan, some areas of western Africa, the Caribbean, and Central viral exposure and challenge. Notably, infants are protected and South America. HTLV-1 infections are also documented from HTLV-1 infection in the early months of life by maternally among immigrant populations and i.v. drug users in the European acquired Ab (15). A vaccine candidate based on an envelope Union and the United States (2). expressing vaccinia virus provides protection to experimentally Despite a robust immune response, HTLV-1 persists in infected challenged primates (16, 17), and an attenuated viral strain individuals throughout life, and high proviral loads are a pre- provides long-term protection against the closely related bovine disposing factor in the progression of HTLV-1–associated dis- leukemia virus (18). These observations suggest that a cost- effective vaccine may be a viable objective for prophylactic Biomedical Research Institute, College of Medicine Dentistry and Nursing, Nine- intervention in HTLV-1–endemic areas. wells Hospital, The University of Dundee, Dundee DD1 9SY, Scotland The viral envelope glycoproteins appear to be good candidates Received for publication January 7, 2011. Accepted for publication April 17, 2011. for a subunit vaccine. The surface glycoprotein (SU) component of This work was supported by project grants (to D.W.B.) from the Association for envelope mediates viral infection of cells by binding to the cellu- International Cancer Research and from Leukaemia and Lymphoma Research and lar entry factors heparan sulfate proteoglycans (19, 20), glucose through pump-priming funds from Tenovus-Scotland. transporter 1 (Glut-1) (21), and neuropilin 1 (22). Subsequently, Address correspondence and reprint requests to Dr. David W. Brighty, Biomedical through a cascade of conformational changes in envelope that Research Institute, College of Medicine Dentistry and Nursing, Ninewells Hospital, The University of Dundee, Dundee DD1 9SY, Scotland. E-mail address: d.w.brighty@ includes isomerization of a SU-transmembrane glycoprotein (TM) dundee.ac.uk intersubunit disulfide (23, 24), envelope catalyzes the fusion of the The online version of this article contains supplemental material. viral and cell membranes allowing entry of the virus into the cell. Abbreviations used in this article: ACD, citric acid, sodium citrate, and dextrose; Neutralizing Abs (10–12, 14) and synthetic inhibitory peptides CTD, C-terminal domain; Glut-1, glucose transporter 1; HOS, human osteosarcoma; block envelope-mediated entry (25–28), suggesting that interfer- HTLV-1, human T cell leukemia virus type 1; HTLV-1pp, HTLV-1 envelope- pseudotyped viral particle; PBST, phosphate buffered saline and 0.5% Tween 20; ing with envelope function can prevent dissemination of viral PMN, polymorphonuclear neutrophil; RBD, receptor-binding domain; rSU, recombi- infection. Although envelope-specific neutralizing Abs are fre- nant SU; SU, surface glycoprotein; TM, transmembrane glycoprotein; tpa, tissue quently observed in natural infections, Abs specifically targeted plasminogen activator. to a functionally critical region of fusion-active envelope fail to Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 antagonize viral entry into cells (29, 30), emphasizing that the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100070 2 HTLV-1 SU-SPECIFIC Abs geometry of native viral envelope and the mode of interaction with ELISA Abs have a profound impact on the ability of Abs to neutralize Microtitre 96-well plates (Nunc; MAXI-Sorp) were coated with anti-human virus infectivity. Understanding the immunogenic properties of IgG (Fc-specific) goat antiserum (Sigma-Aldrich) for 1 h at 37˚C. The HTLV-1 envelope is therefore a critical issue for the development plates were blocked (5% Marvel/PBS/0.2% Tween 20) for 1 h at room of effective vaccines and immunological treatments to combat temperature; sRgp46-Fc–containing Drosophila medium supernatant was HTLV-1 infection. added and incubated for 1 h at room temperature; and unbound SU was removed by washing (53). Subsequently, murine envelope-specific and Although the crystal structure for HTLV-1 SU has not yet been control Abs in hybridoma supernatants or purified mAb at the concen- determined, rigorous examination suggests that the SU is com- trations indicated were added and incubated with the immobilized target prised of two autonomously folding domains separated by Ag for 2 h at room temperature. Plates were washed extensively, and a proline-rich linker peptide (31–36). The N-terminal region, also peroxidase-conjugated anti-mouse IgG (1:10,000 dilution; Sigma-Aldrich) was added and the bound Ab detected using fresh ABTS substrate at 15 known as the receptor-binding domain (RBD), is necessary and mg/ml (in 0.1 M citric acid, 0.06% H2O2). After 10–20 min, the absor- sufficient for binding to Glut-1 on target T cells (21, 33). More- bance was read at 415 nm. Dose-dependent Ab binding was compared by over, transfer of the RBD into a heterologous viral envelope nonlinear regression using GraphPad by Prism, and minima and maxima confers upon the recipient virus the cellular
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