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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

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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 tropism that is typical for each data set were constrained to 0 and 100%, respectively. of HTLV-1 (32, 37). By contrast, the C-terminal domain (CTD) is Syncytium interference assays less well characterized, but accumulating evidence suggests that the CTD not only provides an important structural function in the Syncytium interference assays were performed by standard methods (28, 29, 43, 44). HeLa cells (2 3 105) transfected with the envelope expression envelope trimer, but is also involved in recognition of additional vector pHTE-1 were added to untransfected HeLa target cells (8 3 105). entry factors or coreceptors (38, 39), in determining the differing The effector and target cells were cocultured in the absence or presence of Downloaded from tropisms of HTLV-1 and HTLV-2 (40), and in transducing the the mAbs at the concentrations specified. The cells were incubated for 12– envelope activation signal upon receptor binding (23, 24, 41). To 15 h at 30˚C and returned to 37˚C for 1–2 h, washed twice with PBS, and date, the contribution of these domains to recognition by Ab and fixed in PBS containing 3% paraformaldehyde. Syncytia were stained with Giemsa’s solution (BDH). Assays were performed in triplicate, and the to the sensitivity of HTLV-1 to Ab-mediated neutralization is in- number of syncytia from five fields (3200) per replicate was scored by completely understood. light microscopy.

In this study, we investigate whether a recombinant form of SU http://www.jimmunol.org/ that is competent for receptor binding can induce Ab responses and Pseudotyping assay whether such Abs are capable of inhibiting envelope-mediated Pseudotyped virus was prepared, as described (26, 45). Briefly, 293T cells membrane fusion and viral entry. We demonstrate that many were cotransfected using Genejuice (Novagen) with 10 mg env-defective, anti-SU Abs exhibit some neutralizing activity, whereas some are luciferase-encoding, HIV proviral clone pNL4-3.LUC.R-.E- (46, 47) (obtained through the AIDS Research and Reference Reagent Program, potently neutralizing. The most robustly neutralizing Abs tend to Division of AIDS, National Institute of Allergy and Infectious Diseases, recognize the RBD of SU, but, although the relative binding ef- National Institutes of Health, and donated by N. Landau, New York Uni- ficiency of mAbs may be similar, there are marked differences versity School of Medicine) in the presence or absence of 10 mg pHTE-1. in the inhibitory properties of RBD-targeted Abs. Moreover, we Sodium butyrate (20 mM) was added after 18 h. The sodium butyrate demonstrate that even poorly neutralizing Abs have the ability to medium was changed to normal growth medium after 24 h, and the viral by guest on September 26, 2021 supernatants were harvested after an additional 24-h incubation by cen- recognize HTLV-1 virions and to recruit cytotoxic responses to trifugation at 2500 3 g and filtration through a 0.20-mM micropore filter. HTLV-1–infected cells. Finally, we provide evidence that neu- For transduction of HeLa or HOS cells, triplicate samples of 7 3 105 target tralizing Abs block HTLV-1 infectivity at distinct steps of the viral cells in 1 ml were incubated with 1 ml undiluted or serially diluted virus entry process. We discuss the implications of our findings for stock and plated into wells of a 6-well tissue culture dish. Following overnight incubation at 37˚C/5% CO2, cells were harvested, lysed, and HTLV-1 immunotherapy. assayed for luciferase activity following the manufacturer’s instructions using the Luciferase Assay System (Promega) and a Turner Designs TD- Materials and Methods 20/20 luminometer (Sunnyvale, CA). For Ab inhibition experiments, 1 ml HTLV-1 Env-pseudotyped virus was incubated with 1 ml HeLa or HOS Plasmids and cell lines cells (4 3 105 cells) in the presence or absence of serial dilutions of pu- The plasmids pHTE-1, pMtgp46-Fc have been described (42, 43). HeLa rified mAb. After overnight culture, the cells were lysed and assayed for and human osteosarcoma (HOS) cells were maintained in DMEM sup- luciferase activity using the Promega Luciferase Assay System, as directed plemented with 10% FBS. Sup-T1 cells and primary lymphocytes were by the manufacturer. maintained in RPMI 1640 supplemented with 10% FBS. Insect cells were maintained in Shields and Sang medium supplemented with 10% heat- Flow cytometry inactivated FBS. HTLV-1–infected MT2 and noninfected (control) Sup-T1 cells at a density 6 Expression and purification of sRgp46-Fc proteins of 10 cells/ml were mixed with 0.1 ml cell-free hybridoma culture supernatant or 1 mg/ml purified mAbs and incubated at room temperature for The expression of recombinant sRgp46-Fc protein in insect cells and pu- 1 h with rotation. The cells were pelleted at 1600 rpm for 5 min, in an rification by affinity chromatography on Con A-Sepharose and protein A- Eppendorf C5415C microfuge, washed, and incubated with anti-mouse Sepharose were carried out, as described (43), with the exception that FITC-labeled Ab (1:1000 dilution) in RPMI 1640 medium at room tem- sRgp46-Fc was eluted from protein A-Sepharose using Ab Gentle Elution perature for 1 h in the dark. The cells were washed (PBS/0.1% sodium buffer (Pierce), as recommended by the manufacturer. The concentration azide), fixed (0.5% paraformaldehyde in PBS [pH 7.4]), and kept in of the eluted protein was estimated by Bradford assay, and the recombinant the dark at 4˚C until analyzed by flow cytometery (FACScan; BD Bio- protein was stored at 280˚C in column buffer (20 mm Tris [pH 7.5], 500 sciences). mM NaCl) supplemented with 20% glycerol. Purified sRgp46-Fc was shown to bind to Glut-1–expressing cells, as determined by flow cytometry Microscopy (43). MT2 cells (2 3 106 in 2 ml) were incubated with 250 ml SU mAb hy- Western blotting bridoma culture supernatant at 37˚C/5% CO2 for 20 min and plated onto poly-L-lysine–coated glass coverslips (Sigma-Aldrich). The cells were Western blotting was carried out using standard methods. HTLV-1 envelope incubated at 37˚C/5% CO2 on the coverslips for 60 min. Cells were washed expression was probed using supernatant from murine hybridoma cell lines with PBS, fixed with 3% paraformaldehyde for 15 min at room tempera- producing mAbs raised against sRgp46-Fc, and detected with anti-mouse ture, and then blocked (PBS, containing 10% FBS and 0.25% BSA). The HRP-conjugated secondary Ab at a dilution of 1:2500 in PBS containing cell-bearing coverslips were carefully washed three times in blocking 0.2% (w/v) Marvel and 0.025% Triton X-100. buffer and incubated in the dark with anti-mouse IgG-FITC (1:750; Sigma- The Journal of Immunology 3

Aldrich) at room temperature for 1 h. Coverslips were mounted onto glass proteins’ ability to bind in a dose-dependent and saturable manner slides with Mowiol (Mowiol 4-88; Calbiochem) mounting medium or with to Jurkat and Sup-T1 CD4+ cell lines in flow cytometry assays Vectashield (Vector Laboratories) for differential interference contrast (43). The functional sRgp46-Fc was used to immunize CD1 mice, microscopy. Slides were sealed with nail varnish and left to dry in the dark before examination by fluorescence microscopy and/or differential in- which responded robustly to the immunogen, and subsequently, terference contrast imaging using a Zeiss Axioplan 2 microscope with splenocytes were recovered from three mice with high Ab titres Openlab (Improvision) data acquisition software. (endpoint dilution .1:20,000). Splenocytes from individual mice Isolation of peripheral blood polymorphonuclear neutrophils were fused to SP2/0-Ag14 or, alternatively, NSO-1 myeloma cells and hybridomas selected using standard methods. Over 1700 hy- Buffy coat fractions from healthy donors were obtained from the Scottish bridomas were recovered, of which 168 were positive for acti- Blood Transfusion Service. Polymorphonuclear neutrophils (PMN) were obtained by standard procedures including dextran sedimentation to remove vity to the immunogen. From this group of hybridomas, based on erythrocytes and platelets, hypotonic lysis to remove remaining eryth- the hybridoma fusion partner, mouse donor, Ab isotype, reactivity rocytes and platelets, and Ficoll sedimentation to separate mononuclear with the recombinant Ag, and failure to interact with human IgG, cells from neutrophils. In brief, buffy coat fractions from donors (50 ml 18 independent hybridomas reactive with sRgp46-Fc were selected each) were layered onto citric acid, sodium citrate, and dextrose (ACD) solution with 1 ml ACD for every 5 ml blood. Dextran solution (6% dextran/ for detailed study (Fig. 1A). The selection criteria reduced the 0.9% NaCl) was then added to the ACD/blood mixture. The ACD/dextran/ likelihood that multiple mAbs were derived from the same clonally blood mixture was incubated at room temperature for 45 min, or until expanded splenocyte. Each of the mAbs bound to sRgp46-Fc, but sedimentation was complete. The separated supernatant was centrifuged at did not bind to the control tissue plasminogen activator (tpa)-Fc 1000 rpm (Heraeus; Labofuge 400) for 12 min at 4˚C without break. The fusion product (Fig. 1A), indicating that the binding activity was cell pellet was resuspended in ice-cold ddH2O, and 0.6 M KCl was added directed at the SU component of the envelope-Fc fusion protein. Downloaded from to the cell suspension with 1 ml KCl for every 3 ml cell/H2O suspension. Cells were recovered by centrifugation at 1000 rpm for 6 min at 4˚C Isotyping assays indicated that the panel included six IgG1, six with break, and the process was repeated until no RBCs remained. The IgG2a, five IgG2b, and one IgM Abs (Table I). cell suspension was layered onto Ficoll-Hypaque (density 1.077; Sigma- Representative mAbs were purified from conditioned medium, Aldrich) and centrifuged at 1200 3 g for 30 min at 4˚C, and mononuclear cells were collected at the Ficoll interface, whereas the neutrophils and the relative binding activities were compared in dose- sedimented to the bottom of the Ficoll, where they were recovered. Puri- dependent sRgp46-Fc–binding assays (Fig. 1B). Although these

fied PMN as effector cells were washed with HBSS, enumerated by ELISA-based assays do not allow accurate calculation of disso- http://www.jimmunol.org/ 7 hemocytometry, and resuspended in HBSS/BSA at 2 3 10 cells/ml. Vi- ciation constants (K ) for the Abs, the concentration of Ab re- . . d ability was 99%, and PMNs constituted 90% of leukocytes. PMN were quired to give half-maximal binding can be used as a comparator used immediately after harvest and purification. Respiratory burst assay The Ab-dependent neutrophil respiratory burst was assessed using a chemiluminescence procedure (48). MT2 cells were plated at 200 ml(23 106 cells/ml) per well of a 96-well plate and fixed with 0.05% glutaral- dehyde. The cells were centrifuged onto the plate at 1000 rpm (Heraeus; Labofuge 400) for 10 min, washed with phosphate buffered saline and by guest on September 26, 2021 0.5% Tween 20 (PBST), blocked with 3% BSA/PBST for 1 h at room temperature, and incubated with HTLV-1 SU mAb for 1–2 h or until the purified neutrophils were ready. The MT2 cells were washed once with PBST, PBS, and HBSS/BSA, in turn, and 100 ml luminol (4-amino- phthalhydrazide [Sigma-Aldrich]; 25 3 105 M in HBSS/BSA) was added per well. Subsequently, 2 3 106 neutrophils were added to each well. The cells, in the presence of luminol, were incubated for 10 min at 37˚C/5% CO2 and centrifuged at 1000 rpm for 3 min to precipitate cells. The plate was immediately transferred to the luminometer, and the luminescence of each sample was determined at 1-min intervals over a period of 30 min. Chemiluminescence was recorded as relative light units. The maximal relative light units were determined for each Ab-opsonized sample and control. Maximal relative light values were routinely obtained within 5 min of initiating the assay. Immunoprecipitation of HTLV-1 virions with SU-specific mAbs SU mAb (1 mg) in PBS was incubated with 50 ml mMACS protein A microbeads (Miltenyi Biotec) for 30 min on ice. MT2 cell-free supernatant (1 ml) was subsequently incubated with the Ab-conjugated microbeads for 20 min at room temperature with mixing. The microbeads were captured on m columns (Miltenyi Biotec) and washed with five-column volumes of wash buffer. Intact virions were eluted from the magnetic microbeads following the protocol of the manufacturer. The RNA was extracted from captured virions using TRIzol (Invitrogen) and used for RT-PCR using published RT-PCR primers (49); the remaining eluate was prepared for FIGURE 1. Murine mAbs bind to HTLV-1 SU. A, mAbs derived from analysis of viral protein by Western blotting. immunized mice were examined for reactivity to sRgp46-Fc and the carrier control protein tpa-Fc by ELISA. The Ags (sRgp46-Fc and tpa-Fc) were Results coated onto the wells of a 96-well plate; following incubation with condi- To investigate the immunological properties of HTLV-1 envelope, tioned medium supernatant from each of the hybridomas, the plates were washed and bound Ab was detected. Background reactivity to the control a functional soluble form of recombinant SU (rSU), sRgp46-Fc, 25 308 protein tpa-Fc was subtracted from each of the data points shown. The which includes amino acid residues Ser to Ser of envelope negative control represents the signal obtained from the secondary Ab in the (amino acid coordinates based on the unprocessed envelope pri- absence of a primary SU-specific murine mAb. B, Dose-dependent binding mary translation product), was concentrated and purified by Con of purified mAbs to sRgp46-Fc. mAbs at the concentrations indicated were A and protein A affinity chromatography (43). The receptor- incubated with immobilized sRgp46-Fc. Following washing, bound Ab was binding activity of the purified protein was confirmed by the detected. All data represent the mean 6 SD of triplicate assays. 4 HTLV-1 SU-SPECIFIC Abs

Table I. Isotype and affinity of HTLV-1 SU mAb To begin to characterize the regions of SU recognized by SU- specific mAbs, we compared the reactivity of the mAbs to Hybridoma Isotype and Subclass Half-Maximum Binding (mg/ml) sRgp46-Fc, two envelope deletion constructs, and a control Fc CSK1-9 IgG2a 0.026 protein. These constructs encode SU or its derivatives fused to the CSK2-43 IgG1 ND Fc region of human IgG (Fig. 2A) and are reactive with Fc-specific CSK2-71 IgG1 0.144 control antisera. In ELISA, all mAbs bound to sRgp46-Fc, but not CSK3-13 IgG1 18.40 to a control tpa-Fc product (Fig. 2B). Moreover, mAbs CSK2-71, CSK4-13 IgG2b ND CSK4-24 IgG1 0.016 CSK4-34, CSK11-5, CSK16-13, CSK-N30, and CSK-N37 bound CSK4-34 IgG1 0.222 efficiently to an envelope construct, CTD-Fc, which lacks amino CSK5-37 IgG2a 0.037 acid residues 25–190 of SU (Fig. 2). The region deleted in these CSK7-12 IgG2b 0.501 constructs encodes all of the amino acids currently known to be CSK11-5 IgG2a 0.153 important for binding to the receptor Glut-1 (32). Therefore, CSK11-18 IgG2b 0.075 CSK11-81 IgG2a 0.005 CSK2-71, CSK4-34, CSK11-5, CSK16-13, CSK-N30, and CSK- CSK11-83 IgM ND N37 recognize epitopes that lie in the CTD of SU, in a region that CSK15-18 IgG2a 0.025 is not required for interaction with Glut-1. By contrast, most of the CSK16-13 IgG2b 0.315 remaining mAbs bound only to envelope proteins that included the CSK16-47 IgG2a 0.009 N-terminal receptor-binding domain (RBD-Fc) of SU (residues CSK-N30 IgG2b 0.306 25 190 CSK-N37 IgG1 0.275 Ser to Leu ), but did not bind to envelope fragments that lack these sequences (Fig. 2), indicating that the majority of the mAbs Downloaded from recognize the RBD of SU. Only CSK2-43 bound to both the RBD- of relative binding affinity. Analysis of the binding curves and Fc and CTD-Fc proteins, but did not bind to tpa-Fc, indicating that calculation by nonlinear regression analysis of the Ab concen- this Ab most likely recognizes an epitope that overlaps the junc- tration required to give half-maximal binding (Table I) indicated tion of the RBD and CTD (Fig. 2B). In addition, each mAb was that there is a ∼50-fold difference in the relative binding activity examined for recognition of denatured Ag by Western blot anal-

within the main group of mAbs. Three Abs, CSK1-9, CSK16-47, ysis against sRgp46-Fc, rSU (without the Fc-tag), and envelope- http://www.jimmunol.org/ and CSK11-81, exhibit high relative binding to sRgp46-Fc, derived fragments (Fig. 2C). Although raised against intact func- whereas mAb CSK3-13 demonstrated the lowest binding activ- tional Ag, all of the mAbs displayed efﬁcient binding to de- ity (Fig. 1B, Table I). natured envelope, including the untagged SU. Therefore, the Abs by guest on September 26, 2021

FIGURE 2. Mapping of SU domains recognized by mAbs. The constructs used to map regions of SU recognized by mAbs are shown. A, Each construct is preceded by the signal sequence from human tpa to aid secretion, and the boxes represent HTLV-1 envelope sequences; sRgp46-Fc includes amino acid residues Ser25 to Ser308 of envelope, RBD-Fc includes amino acids Ser25 to Leu190, and CTD-Fc includes residues Leu190–Ser308, and each construct is fused to the Fc region of human IgG; the thin line represents deleted amino acid residues. B, Each mAb was examined for reactivity to sRgp46-Fc, or its truncated derivatives CTD-Fc and RBD-Fc, or the control protein tpa-Fc by ELISA. The Ags (sRgp46-Fc, CTD-Fc, RBD-Fc, and tpa-Fc) were coated onto the wells of a 96-well plate; following incubation with conditioned cell-free medium supernatant from each of the hybridomas, the plates were washed and bound Ab was detected. A control mAb specific to the Fc region of human IgG was included as a control. C, Each mAb was examined for recognition of denatured sRgp46-Fc, or its truncated derivatives CTD-Fc and RBD-Fc, the control protein tpa-Fc, or untagged rSU by Western blotting. Conditioned medium supernatants from untransfected S2 cells were included as negative controls for SU specificity. Typical results for a representative sample of the mAbs are shown. The reactivity of the remaining mAbs are given in Table II. The Journal of Immunology 5 are not dependent on the conformation of the Ag for efficient for these results is that the epitopes recognized by these Abs are binding and most likely recognize linear epitopes of SU. The masked or occluded on the intact viral envelope complex and that Western analysis also confirmed the domain specificity of each removal of SU from TM, or denaturation of SU, increases the mAb. exposure of these epitopes. However, Env has been observed to Candidate immunogens for subunit vaccines must routinely and interact with neuropilin 1 and Glut-1 in HTLV-1–infected cells robustly elicit Abs that recognize the native, extensively glyco- (21, 22); consequently, an alternative scenario is that such in- sylated, and trimeric structures of viral envelope. To explore the teractions may prevent binding of these mAbs to envelope on reactivity of anti-SU mAbs with virally expressed envelope, several infected cells. Although useful in Western analysis, due to their complementary assays were employed. First, binding of each mAb lack of reactivity with native viral envelope, mAbs CSK2-43, to HTLV-1–infected MT2 cells was examined by flow cytome- CSK3-13, and CSK11-83 were not studied further. Generally, try. The majority of the mAbs bound efficiently to the HTLV-1– mAbs that displayed strong relative binding to sRgp46-Fc also infected cells (Fig. 3A,3B), whereas none of the mAbs bound to exhibit robust binding to infected T cells. the control Sup-T1 cells. These results indicate that the majority To visually confirm binding of mAbs to viral envelope, chron- of the mAbs raised against sRgp46-Fc recognize viral envelope ically infected MT2 cells were probed with SU-specific mAbs and displayed on the surface of infected human T cells. By contrast, examined by fluorescence microscopy. The majority of mAbs that several mAbs, CSK2-43, CSK3-13, and CSK11-83, bound poorly were reactive with envelope in flow cytometry studies also dem- to HTLV-1–infected cells (Fig. 3B), but bound effectively to onstrated binding to viral envelope by fluorescence imaging. sRgp46-Fc (Figs. 1, 2) and recognize SU by Western blotting (data Typically, mAbs exhibit homogenous staining around the plasma not shown). The simplest and, in our view, most likely explanation membrane of unstimulated MT2 cells (Fig. 3C) consistent with the Downloaded from expected localization of viral envelope. No staining was observed with uninfected T cell lines by either fluorescence microscopy or flow cytometry (Supplemental Fig. 1), reinforcing the view that the cell staining observed on MT2 cells is due to specific recognition of the SU component of the viral envelope glycoprotein

complex. http://www.jimmunol.org/ Ab-dependent neutralization of envelope-mediated membrane fusion and viral entry Patient sera and mAbs derived from infected individuals interfere with viral infection of cells, and this neutralizing activity is primarily directed at viral envelope. We therefore examined the murine mAbs for the ability to block binding of SU to cells, envelope-mediated membrane fusion, and viral entry into cells. First, the ability of the mAbs to block binding of SU to uninfected by guest on September 26, 2021 T cells was examined by flow cytometry (Fig. 4). Many, but not all, of the mAbs were effective inhibitors of SU binding to uninfected Sup-T1 cells. In particular, CSK1-9, CSK7-12, and CSK11-18 were all potent inhibitors of SU binding, whereas CSK4-24, CSK4-34, CSK11-81, CSK16-47, CSKN-30, and CSKN-37 exhibit modest inhibition of SU binding, and the remaining mAbs show little, if any, inhibitory activity. Thus, one potential and predictable mechanism by which these Abs inhibit HTLV-1 viral entry is via direct steric occlusion of receptor binding. Surpris- ingly, a few mAbs, typified by CSK11-83, appear to enhance binding of sRgp46-Fc to cells. It is not yet known how these mAbs promote binding of soluble SU to cells, but this observation is discussed in more detail below. Based on the above data, a group of the mAbs typical of the FIGURE 3. Murine mAbs recognize HTLV-1 envelope displayed on various Ab classes and RBD or CTD specificities was selected for infected cells. A, Chronically infected MT2 cells were incubated with anti-SU mAbs, or irrelevant control mAb (anti-Fc), as indicated (open histograms), or high-yield purification and examined for the ability to inhibit control MT2 cells probed in the absence of primary Ab (solid histogram). membrane fusion in syncytium interference assays (Fig. 5A). HeLa Bound Ab was detected using FITC-conjugated anti-murine IgG antisera and cells transfected with the subgenomic HTLV-1 envelope expres- flow cytometry. Typical results are illustrated for Abs CSK1-9, CSK3-13, sion construct pHTE-1 were cocultured with nontransfected HeLa CSK4-24, and CSK11-81. No binding was observed with uninfected Sup-T1 target cells in the presence of purified mAb. In the absence of Ab, cells (data are shown in Supplemental Fig. 1). B, All mAbs were examined or in the presence of irrelevant anti–HIV-1 gp120 control mAb for the ability to bind to MT2 cells, as described above, and the results dis- (178.1), extensive membrane fusion and syncytia production were played as mean fluorescence intensity of the cell population after subtraction observed (Fig. 5A,5B). By contrast, several of the anti-SU mAbs, of the background signal produced in the presence of secondary Ab alone for example CSK7-12, CSK11-18, CSK4-24, and CSK1-9, effi- (typical data from one of multiple independent assays [n =3]areshown).C, ciently blocked syncytium formation, as observed by low-power Typical immunofluorescence images (original magnification 3630) of HTLV- 1–infected MT2 cells probed with the anti-SU mAbs CSK1-9, CSK4-24, microscopy (Fig. 5A,5B). Although CSK7-12 has modest relative CSK11-5, and CSK16-47. These images are typical of those obtained with all binding activity to gp46 (Fig. 1B, Table I), mAbs CSK11-18, MT2 immunofluorescence-positive mAbs (Table II). No binding was ob- CSK4-24, and CSK1-9 exhibit strong relative binding to rSU, served with uninfected Sup-T1 cells by microscopy or by flow cytometry (see and all of these mAbs efficiently recognize viral envelope in flow Supplemental Fig. 1). cytometry assays. In contrast, some mAbs, CSK4-34, CSK15-18, 6 HTLV-1 SU-SPECIFIC Abs Downloaded from

FIGURE 4. Some mAbs efficiently block binding of sRgp46-Fc to hu- http://www.jimmunol.org/ man T cells. Sup-T1 cells were incubated with sRgp46-Fc in the presence or absence of anti-SU mAbs, or in the presence of an irrelevant anti-murine Fc Ab. Subsequently, cells were washed and bound sRgp46-Fc was probed with FITC-conjugated anti-human Fc Ab and detected by flow cytometry. A, Typical binding data are shown. Solid histogram, untreated Sup-T1 cells; open red histogram, Sup-T1 cells in the presence of sRgp46-Fc and binding detected with anti-Fc FITC-labeled Ab and in the absence of anti- SU mAb; open green histogram, Sup-T1 cells in the presence of sRgp46- Fc and the indicated SU-specific murine mAb. In the top left control panel, by guest on September 26, 2021 the green histogram represents Sup-T1 cells in the presence of anti-Fc FITC-labeled Ab, but in the absence of sRgp46-Fc. B, All mAbs were examined for the ability to inhibit binding of sRgp46-Fc to Sup-T1 cells by FIGURE 5. Many SU-specific mAbs display neutralizing activity. The the method described above. Data are plotted as percentage of inhibition of ability of murine mAbs to inhibit Env-dependent membrane fusion was maximal fluorescence relative to cells probed in the absence of SU-specific examined in syncytium interference assays. A, HeLa target cells were co- mAbs. All data values represent the mean of triplicate samples. cultured with Env-expressing HeLa cells in the presence of 10 mg/ml mAb, as indicated. Inhibition of syncytium formation was examined by light microscopy of Giemsa-stained monolayers; the data are displayed as per- and CSK16-47, antagonized membrane fusion weakly even at centage of inhibition of syncytium formation relative to untreated controls. B, high mAb concentrations. Notably, although CSK16-47 binds to Typical Giemsa-stained monolayers for control cells in the absence of en- the RBD of SU, is among the Abs with highest relative binding velope expression (Control) and in envelope-expressing cells in the absence (No mAb) or presence of purified mAbs CSK1-9, CSK4-24, and CSK7-12 or activity to monomeric SU, and very efficiently binds to virally the irrelevant control Ab 178.1 (HIV-1 gp120 specific) are illustrated. C,The infected cells, it is a relatively weak inhibitor of syncytium for- most robustly neutralizing Abs CSK1-9, CSK4-24, CSK7-12, and CSK11-18 mation even at high Ab concentrations (Fig. 5A). By comparison, were compared in dose-dependent syncytium interference assays. Briefly, the most potently neutralizing Abs within this panel, CSK7-12, target and effector cells were mixed in the presence of increasing doses of CSK11-18, CSK4-24, and CSK1-9, also recognize the RBD of mAb, and the percentage of inhibition of syncytium formation was de- SU. Therefore, mAbs CSK7-12, CSK11-18, CSK4-24, and CSK1- termined. All assays were performed in triplicate, and the number of syncytia 9 were compared directly for inhibition of membrane fusion (Fig. from five fields (original magnification 3200) per replicate was scored by 5C). In each case, the Abs displayed potent dose-dependent in- light microscopy. hibition of membrane fusion, and CSK7-12, an Ab with modest relative binding activity for SU, was consistently the most effective inhibitor in these assays (Fig. 5C). By contrast, CSK1-9, blocked entry of HTLV-1 envelope-pseudotyped viral particles a mAb with high relative binding affinity, was less effective than (HTLV-1pp). Robust transduction of luciferase was observed in the CSK7-12 (Fig. 5). Therefore, the ability to inhibit membrane fu- absence of Ab or, alternatively, in the presence of increasing doses sion does not correlate strictly either with the efficiency of binding of an irrelevant control Ab. By contrast, a strong and dose- to envelope or recognition of the RBD. dependent inhibition of HTLV-1pp entry and luciferase trans- To examine the ability of the mAbs to block envelope-mediated duction was observed for mAbs CSK7-12, CSK4-24, and CSK1-9 entry of free viral particles, envelope-deficient HIV-based lucifer- (Fig. 6). Again, inhibition of HTLV-1pp entry did not correlate ase-transducing viral particles were pseudotyped with HTLV-1 strictly with Ab affinity; although CSK1-9 is one of the strongest envelope and used in interference of infectivity assays. Each of binding Abs, it did not inhibit as potently as CSK7-12, especially the mAbs that efficiently blocked syncytium formation also at low Ab concentrations. The Journal of Immunology 7

nescence-based in vitro assay (48). Samples of HTLV-1–infected MT2 cells were opsonized with each of the independent SU- specific mAbs and dispensed into 96-well plates. Subsequently, purified PMN and luminol were added, and the plates were immediately assayed for activation of the PMN respiratory burst, as determined by hydrogen peroxide production and luminol turnover detected as light emission. Maximal relative light output developed within 5 min of PMN addition and decayed over time. PMN incubated with the uninfected T cell line Sup-T1 did not exhibit activation of the respiratory burst (Fig. 8). However, even in the absence of Ab, a very low, but reproducible level of activation of the PMN respiratory burst was noted in the presence of FIGURE 6. Anti-SU–specific mAbs neutralize viral infectivity. Murine HTLV-1–infected T cells. By contrast, MT2 cells opsonized with mAbs (as indicated) were incubated with uninfected Sup-T1 cells in the anti-SU mAbs markedly enhanced the PMN respiratory burst (Fig. presence of HIV-1 luciferase-transducing viral particles pseudotyped with 8) and, significantly, both robustly and weakly neutralizing Abs HTLV-1 envelope. Following infection and recovery, the cells were assayed for transduced luciferase activity and the data were plotted as per- induced strong responses in these assays. In particular, CSK11-18 centage of inhibition relative to untreated viral particles. and CSK16-47 were just as effective at stimulating a PMN respiratory burst despite the fact that CSK11-18 is one of the more

robustly neutralizing Abs and CSK16-47 is only weakly neutral- Downloaded from Both neutralizing and nonneutralizing mAbs bind to viral izing (Table II). Thus, the ability to recruit and activate immune particles effector cells is not tightly coupled to the neutralizing proficiency It was intriguing that despite recognizing SU and interacting with of HTLV-specific Abs. envelope on the surface of infected cells, some mAbs are unable to inhibit membrane fusion or neutralize the entry of HTLV-1pp. A Discussion plausible explanation for the lack of neutralizing activity of some HTLV-1 infections induce adaptive immune responses to viral Ag, http://www.jimmunol.org/ SU-reactive mAbs is that, contrary to neutralizing mAbs, non- but, despite robust immune activation, infected individuals fail to neutralizing mAbs do not bind effectively to trimeric envelope clear virus and virally infected cells. The accumulating data suggest displayed on the surface of cells or virions. We therefore tested that there is an ongoing war of attrition between the immune system the ability of two neutralizing mAbs (CSK1-9 and CSK11-81), and the virus, wherein the immune response acts to eradicate the one weakly neutralizing (CSK16-47), and one nonneutralizing virus and the virus employs diverse, but inadequately understood (CSK16-13) mAb of similar relative binding affinity to immuno- mechanisms to avoid immune surveillance. A clear view of the precipitate HTLV-1 particles produced by chronically infected immunological pathways and viral targets required for successful MT2 cells. Surprisingly, both neutralizing and poorly neutralizing suppression or eradication of HTLV-1 replication is critical to the mAbs were effective at capturing virus particles (Fig. 7), indicating development of vaccines and novel immunological strategies to by guest on September 26, 2021 that the poor neutralizing activity is not associated with a failure to combat HTLV-1–associated disease. As a step toward this objec- recognize viral envelope on the virion surface. tive, we in this study demonstrate that a recombinant HTLV-1 Ab-dependent recruit and activation of immune effector cells to envelope-derived immunogen displays properties of value to sub- HTLV-infected cells unit vaccine design and induces robust humoral immune responses in mice. The immunological properties of sRgp46-Fc Direct neutralization of virus by envelope-specific Ab is important for prevention of de novo viral infection of cells, but, in addition, Ab effector functions contribute significantly to a robust immune response. We therefore sought to test whether neutralizing and nonneutralizing mAbs produced in response to vaccination with a soluble recombinant Ag can mobilize cell-mediated immune responses to HTLV-1–infected cells. Each of the isolated mAbs was examined for the ability to enhance the Ab-dependent cytotoxic respiratory burst of PMN using an established chemilumi-

FIGURE 8. Anti-SU mAbs recruit PMN to HTLV-1–infected cells and activate the neutrophil respiratory burst. HTLV-1–infected MT2 cells were opsonized with each of the indicated anti-SU mAbs and mixed with freshly FIGURE 7. Murine mAbs with markedly differing neutralizing activi- isolated human neutrophils. The PMN respiratory burst was assayed by ties recognize envelope displayed on HTLV-1 virions. Virions from cell- standard procedures using a luminol turnover method (48). The respiratory free MT2 supernatants were immunoprecipitated with the anti-SU mono- burst of neutrophils, shown as maximal relative light units, in the presence clonals CSK1-9, 11-81, 16-13, and 16-47, and the irrelevant HIV-1 gp120- of opsonized MT2 cells, was compared with neutrophils incubated in the speciﬁc control Ab 178.1, or in the absence of Ab. Captured viral particles presence of MT2 cells opsonized by an irrelevant anti-murine Fc mAb, were processed and analyzed for genomic RNA by RT-PCR and gel anti-Fc; or with MT2 cells in the absence of Ab, PMN + MT2; or neu- electrophoresis (top panel; control = template-free RT-PCR reaction), or trophils in the presence of uninfected Sup-T1 cells, PMN + Sup-T1; or for viral capsid protein P19 content by Western blotting (lower panel). unstimulated neutrophils, PMN. 8 HTLV-1 SU-SPECIFIC Abs

Table II. Summary of mAb properties

Inhibition of Inhibition of Inhibition of mAb Isotype Region Immunoﬂuorescence SU Binding Syncytium Formation Virus Entry CSK1-9 IgG2a RBD +++ ++++ +++ +++ CSK2-43 IgG1 CTD + 2 ND ND CSK2-71 IgG1 CTD + 2 ND ND CSK3-13 IgG1 RBD 22ND ND CSK4-13 IgG2b RBD 2 +NDND CSK4-24 IgG1 RBD +++ ++ +++ +++ CSK4-34 IgG1 CTD +++ ++ + 2 CSK5-37 IgG2a RBD 22ND ND CSK7-12 IgG2b RBD +++ ++++ ++++ +++ CSK11-5 IgG2a CTD +++ 2 ND ND CSK11-18 IgG2b RBD ++ ++++ +++ +++ CSK11-81 IgG2a RBD +++ +++ ++ ND CSK11-83 IgM RBD ND 2 ND ND CSK15-18 IgG2a RBD 22 2ND CSK16-13 IgG2b CTD +++ 22ND CSK16-47 IgG2a RBD +++ +++ + ND CSK-N30 IgG1 CTD +++ ++ + 2

CSK-N37 IgG2b CTD +++ ++ ND ND Downloaded from ++++, very strong activity; +++, strong activity; ++, moderate activity; +, weak activity; 2, no activity; ND, not determined. have enabled the selection of a diverse panel of SU-speciﬁc mAbs block binding of the SU to cell surface receptors, whereas others that will facilitate analysis of the immunological features of en- (CSK15-18) block fusion at a postreceptor-binding step of entry

velope. (Figs. 4, 5). Interestingly, based on the observation that the neu- http://www.jimmunol.org/ Significantly, the murine mAbs exhibit properties that are typical tralizing proficiency exceeds the ability of the Ab to prevent SU of Abs produced during natural infections. The mAbs bind to binding to receptor, we find that mAbs such as CSK4-24 appear to virally expressed envelope displayed both on infected cells and, interfere with both receptor binding and the postbinding events of most importantly, on virion surfaces. The majority of the mAbs entry. To our knowledge, this is the first data to successfully re- possess some neutralizing activity, and a few are robustly neu- solve the ability of Abs to neutralize HTLV-1 entry by disrupting tralizing in assays of membrane fusion and pseudovirus entry. The independent steps of the binding, fusion, and entry process. neutralizing activity of mAbs in syncytium interference assays is Alkylation studies coupled with elegant cryo-electron micros- particularly pertinent to natural infections because much of the copy (23, 24, 41, 53) demonstrate that, upon receptor binding, the viral dissemination in infected individuals occurs by transfer of envelope protein of MLV undergoes a radical change in confor- by guest on September 26, 2021 virus between cells across regions of close cell-to-cell contact (50, mation. Receptor binding induces an isomerization and disruption 51). Syncytium formation may therefore be viewed as an extreme of the SU-TM intersubunit thiol, which triggers the reorganization outcome of envelope-mediated processes that naturally occur of the SU subunits to form an open ring-shaped structure. It has within regions of close membrane apposition. Notably, the most been suggested that the TM subunit may extend through the center effective neutralizing activity is associated with Abs that recog- of the trimeric SU complex to induce membrane fusion (41). If nize the RBD of SU. Nevertheless, despite similar relative binding this is indeed the case, the critical fusion-active surfaces of TM are efficiencies, there are marked differences in the neutralizing pro- likely to be sterically shielded from neutralizing Ab due to the ficiencies of RBD-specific mAbs, indicating that not only affinity, presence of the SU subunits and the proximity of the viral and cell but also epitope recognition determines the neutralizing properties membranes. By comparison, SU subunits are likely to have sur- of anti–HTLV-1 Abs. Furthermore, some poorly neutralizing Abs faces exposed to Ab in the prefusogenic state and following exhibit efficient recognition of HTLV-1–infected cells; perhaps, binding of SU to cell surface receptors. Our observations that such Abs recognize alternative forms of envelope that are not some SU-specific Abs block the postreceptor-binding steps of compatible with membrane fusion and viral entry (discussed in entry indicate that such mAbs may interfere with coreceptor or detail below). cofactor recruitment (38, 39) or, alternatively, may prevent the The neutralizing properties of the HTLV-1 SU-specific mAbs receptor-induced changes in SU conformation that are required to contrast strikingly with the activity of mAbs previously raised activate the fusogenic properties of envelope. Understanding the against the fusion-associated structures of TM (29, 30, 52). Despite molecular mechanism by which these mAbs neutralize viral entry the ability of anti-TM mAbs to block binding of leash and helical will provide greater insight into envelope function and the im- region-derived peptides to the coiled coil in vitro, to recognize cell munology of HTLV-1 infections. surface displayed viral envelope, and to target a region critical to A perplexing aspect of these studies is that despite efficient the membrane fusion process, these Abs unanimously failed to recognition of the SU on infected cells and the ability to bind to inhibit either envelope-mediated membrane fusion or viral entry envelope on viral particles, some mAbs are poor inhibitors of into cells (29, 30, 52). By contrast, most of the mAbs targeted to membrane fusion and viral entry. It is difficult to reconcile binding SU exhibit some level of neutralizing activity. The collated data of a large Ab (150 kDa) to a small surface glycoprotein (46 kDa) suggest that in the fusion-active envelope structure, the coiled coil without loss of protein function. Indeed, for HIV-1, a compel- of TM is poorly accessible to Ab and is therefore protected from ling argument has been made that if an Ab binds to the wild- the neutralizing activity of TM-targeted mAbs. Conversely, SU type envelope complex, it will neutralize (54–57). Consistent with appears to be sensitive to neutralization throughout the receptor- this notion, a heterologous Ab can inhibit and neutralize HIV-1 binding and entry process. In particular, our data demonstrate infectivity when the epitope recognized by that Ab is engineered that some mAbs (CSK1-9, CSK7-12, CSK11-18) effectively into the gp120 subunit of HIV-1 envelope (56, 57). By contrast, The Journal of Immunology 9 current models of Ab activity against hepatitis C virus suggest that HIV-1, pose unique technical challenges and have often suffered neutralizing activity is confined to Abs that bind to a limited set of disappointing setbacks. Nevertheless, HTLV-1 is an attractive target functionally important epitopes on the viral E2 envelope protein for vaccine development. The HTLV-1 envelope glycoprotein shows (58, 59). Such differences in neutralizing activity may reside in the relatively little sequence variation in and between infected individuals precise and mutually distinct arrangement of the envelope com- (65); envelope is a primary target for humoral and cell-based immune plexes of these highly divergent viruses. For HTLV-1, it remains to responses in natural infections; an attenuated strain of the related be determined whether binding of an Ab to the envelope complex bovine leukemia virus induces sustained resistance to experimental is compatible with envelope function, or whether Ab must be bovine leukemia virus challenge (18); an envelope-based HTLV-1 bound to a restricted set of critical epitopes for neutralization to immunogen provides protection in primate models of HTLV-1 in- occur. Previously, we have provided evidence that nonfunctional fection (16, 17); and finally, infants born to HTLV-1–infected mothers forms of envelope exist on the surface of infected cells and that are protected from HTLV-1 infection in the first months of life by fusion-incompetent forms of envelope may account for the bind- maternally acquired Abs (15). Collectively, these observations sug- ing of nonneutralizing Ab to envelope-expressing cells (29, 30). gest that an effective envelope-based subunit vaccine is an achievable The ability of nonneutralizing or poorly neutralizing Ab to bind to objective for therapeutic intervention in HTLV-1 infections. virus particles may reflect that nonfunctional forms of envelope There are significant challenges to address before an effective are also found on virions. If this proves to be the case, the display HTLV-1 vaccine can be realized. For example, the native pre- of nonfunctional envelope structures may act as decoy Ags that fusogenic structures and fusion-active forms of envelope may both are used by the virus to confound and evade an effective immune be targeted by Ab, but it is not clear which of these forms is most response. Further studies are required to test these views. appropriate to vaccine design. Moreover, it is probable that a tri- Downloaded from Intriguingly, a few mAbs, for example CSK11-83 (Fig. 5B), meric form of envelope would produce a more effective, longer- weakly, but consistently enhance binding of HTLV-1 SU to cells. lasting response than a monomeric subunit approach, but this At present, we do not know how this is achieved, but we suspect awaits experimental validation. Our study provides information and that such Abs lock the soluble SU in a particular conformation that insight into the way that HTLV-1 envelope interacts with the hu- is primed and highly amenable to receptor binding. In view of this moral immune response and how these interactions may be im-

possibility, it should be noted that CSK11-83 binds well to sRgp46- portant for viral pathogenesis and ultimately for future vaccine http://www.jimmunol.org/ Fc, but poorly to native trimeric envelope displayed on the surface initiatives. A successful vaccine strategy for HTLV-1 would have of infected cells. It may be that mAbs such as CSK11-83 recognize considerable clinical impact and could act as a useful pathfinder for surfaces of sRgp46 that are normally buried at the interface of the the more intractable and highly mutable viruses such as HIV-1. SU subunits within the envelope trimer; in binding to these surfaces, the Ab may provide additional structural rigidity to the soluble SU. These ideas will be tested in future fine-mapping studies. Acknowledgments A significant finding of our study is that both neutralizing and We thank Lisa Orram for assistance with the purification of Abs, Dr. Kulpash weakly neutralizing Abs recruit and robustly activate the cytotoxic Nurkiyanova for technical assistance with hybridoma fusion, Dr. Abdenour responses of neutrophils. A recent study also demonstrated that Soufi for independent confirmation of results, Dr. Daniel Lamb and mem- by guest on September 26, 2021 bers of the laboratory for useful discussions, and the East Scotland Blood nonneutralizing Abs are capable of recruiting complement to en- Transfusion Services for supplying leukopacks. We also thank Dr. Jenny velope and envelope-expressing cells (29). Our observations in- Woof and Dr. Lindsay Tulloch for helpful comments on the manuscript. dicate that, in addition to neutralizing activity, Ab effector function is likely to play a significant role in controlling HTLV-1 dissemination and viral burden during natural infections. Ab-dependent Disclosures cellular cytotoxicity may be of particular benefit in suppression of The authors have no financial conflicts of interest. HTLV-1 and HTLV-1–associated disease as the vast majority of de novo viral infections occur by direct transfer of virus from infected cell to target cell through sites of intimate cellular contact, known References as the virological synapse, rather than through infection by free 1. Cann, A. J., and I. S. Y. Chen. 1996. Human T-cell leukaemia virus types I and II. In Fields Virology, 3rd Ed. Lippincott-Raven, Philadelphia, p. 1849–1880. viral particles (50). Due to the close contact of the infected cell 2. Anonymous. 1996. Human T-cell lymphotropic viruses. In Human Immunode- and uninfected cell membranes during cell-to-cell transmission, ficiency Viruses and Human T-Cell Lymphotropic Viruses. I. W. Group, ed. IARC Monographs, IARC, Lyon, France, p. 261–390. HTLV-1 may be protected from the neutralizing properties of Ab, 3. Hanon, E., R. E. Asquith, G. P. Taylor, Y. Tanaka, J. N. Weber, and but, because of cell surface display of envelope, infected cells C. R. Bangham. 2000. High frequency of viral protein expression in human should remain sensitive and vulnerable to Ab-dependent cellular T cell lymphotropic virus type 1-infected peripheral blood mononuclear cells. AIDS Res. Hum. Retroviruses 16: 1711–1715. cytotoxicity and Ab-mediated complement fixation. Nonetheless, 4. Asquith, B., E. Hanon, G. P. Taylor, and C. R. Bangham. 2000. Is human T-cell despite compelling evidence that effector function has a profound lymphotropic virus type I really silent? Philos. Trans. R. Soc. Lond. B Biol. Sci. impact on the success of passive immunotherapy for HIV-1, for 355: 1013–1019. 5. Hanon, E., J. C. Stinchcombe, M. Saito, B. E. Asquith, G. P. Taylor, Y. Tanaka, other viral infections, and for treatment of solid tumors (60, 61), J. N. Weber, G. M. Griffiths, and C. R. Bangham. 2000. Fratricide among CD8 the role of Ab effector function has been largely neglected in (+) T lymphocytes naturally infected with human T cell lymphotropic virus type I. Immunity 13: 657–664. studies of immune control and pathogenesis of HTLV-1 and other 6. Bangham, C. R. 2003. The immune control and cell-to-cell spread of human viral infections (61). Although clearly important, neutralizing pro- T-lymphotropic virus type 1. J. Gen. Virol. 84: 3177–3189. ficiency is only one aspect of Ab function that should be con- 7. Yamano, Y., M. Nagai, M. Brennan, C. A. Mora, S. S. Soldan, U. Tomaru, N. Takenouchi, S. Izumo, M. Osame, and S. Jacobson. 2002. 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Human monoclonal antibody directed against an envelope 10 HTLV-1 SU-SPECIFIC Abs

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