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of June 16, 2017.
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
Supplementary
Material
http://www.jimmunol.org/content/suppl/2011/06/06/jimmunol.110007
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Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 6 June 2011
http://www.jimmunol.org/content/early/2011/06/06/jimmun
ol.1100070
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
H
uman T cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that in some infected individuals causes
an aggressive adult T cell leukemia/lymphoma or a
progressive demyelinating disease known as HTLV-1–associated
myelopathy/tropical spastic paraparesis (1, 2). HTLV-1 infections
are widespread. The virus is prevalent in the Kyushu prefecture of
Japan, some areas of western Africa, the Caribbean, and Central
and South America. HTLV-1 infections are also documented
among immigrant populations and i.v. drug users in the European
Union and the United States (2).
Despite a robust immune response, HTLV-1 persists in infected
individuals throughout life, and high proviral loads are a predisposing factor in the progression of HTLV-1–associated disBiomedical Research Institute, College of Medicine Dentistry and Nursing, Ninewells Hospital, The University of Dundee, Dundee DD1 9SY, Scotland
Received for publication January 7, 2011. Accepted for publication April 17, 2011.
This work was supported by project grants (to D.W.B.) from the Association for
International Cancer Research and from Leukaemia and Lymphoma Research and
through pump-priming funds from Tenovus-Scotland.
Address correspondence and reprint requests to Dr. David W. Brighty, Biomedical
Research Institute, College of Medicine Dentistry and Nursing, Ninewells Hospital,
The University of Dundee, Dundee DD1 9SY, Scotland. E-mail address: d.w.brighty@
dundee.ac.uk
The online version of this article contains supplemental material.
Abbreviations used in this article: ACD, citric acid, sodium citrate, and dextrose;
CTD, C-terminal domain; Glut-1, glucose transporter 1; HOS, human osteosarcoma;
HTLV-1, human T cell leukemia virus type 1; HTLV-1pp, HTLV-1 envelopepseudotyped viral particle; PBST, phosphate buffered saline and 0.5% Tween 20;
PMN, polymorphonuclear neutrophil; RBD, receptor-binding domain; rSU, recombinant SU; SU, surface glycoprotein; TM, transmembrane glycoprotein; tpa, tissue
plasminogen activator.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100070
ease (3, 4). Nonetheless, cytotoxic T cell (CTL) (5–8) and
HTLV-1–specific B cell expansion (9–14) contribute to antiviral
activity, restrict viral dissemination, and help determine proviral
load. Neutralizing Abs are often observed in HTLV-1 infections
(10–12, 14), and accumulating evidence suggests that a primed
immune response can be protective or prevent infection postviral exposure and challenge. Notably, infants are protected
from HTLV-1 infection in the early months of life by maternally
acquired Ab (15). A vaccine candidate based on an envelope
expressing vaccinia virus provides protection to experimentally
challenged primates (16, 17), and an attenuated viral strain
provides long-term protection against the closely related bovine
leukemia virus (18). These observations suggest that a costeffective vaccine may be a viable objective for prophylactic
intervention in HTLV-1–endemic areas.
The viral envelope glycoproteins appear to be good candidates
for a subunit vaccine. The surface glycoprotein (SU) component of
envelope mediates viral infection of cells by binding to the cellular entry factors heparan sulfate proteoglycans (19, 20), glucose
transporter 1 (Glut-1) (21), and neuropilin 1 (22). Subsequently,
through a cascade of conformational changes in envelope that
includes isomerization of a SU-transmembrane glycoprotein (TM)
intersubunit disulfide (23, 24), envelope catalyzes the fusion of the
viral and cell membranes allowing entry of the virus into the cell.
Neutralizing Abs (10–12, 14) and synthetic inhibitory peptides
block envelope-mediated entry (25–28), suggesting that interfering with envelope function can prevent dissemination of viral
infection. Although envelope-specific neutralizing Abs are frequently observed in natural infections, Abs specifically targeted
to a functionally critical region of fusion-active envelope fail to
antagonize viral entry into cells (29, 30), emphasizing that the
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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
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
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.
2
Materials and Methods
Plasmids and cell lines
The plasmids pHTE-1, pMtgp46-Fc have been described (42, 43). HeLa
and human osteosarcoma (HOS) cells were maintained in DMEM supplemented with 10% FBS. Sup-T1 cells and primary lymphocytes were
maintained in RPMI 1640 supplemented with 10% FBS. Insect cells were
maintained in Shields and Sang medium supplemented with 10% heatinactivated FBS.
Expression and purification of sRgp46-Fc proteins
The expression of recombinant sRgp46-Fc protein in insect cells and purification by affinity chromatography on Con A-Sepharose and protein ASepharose were carried out, as described (43), with the exception that
sRgp46-Fc was eluted from protein A-Sepharose using Ab Gentle Elution
buffer (Pierce), as recommended by the manufacturer. The concentration
of the eluted protein was estimated by Bradford assay, and the recombinant
protein was stored at 280˚C in column buffer (20 mm Tris [pH 7.5], 500
mM NaCl) supplemented with 20% glycerol. Purified sRgp46-Fc was
shown to bind to Glut-1–expressing cells, as determined by flow cytometry
(43).
Western blotting
Western blotting was carried out using standard methods. HTLV-1 envelope
expression was probed using supernatant from murine hybridoma cell lines
producing mAbs raised against sRgp46-Fc, and detected with anti-mouse
HRP-conjugated secondary Ab at a dilution of 1:2500 in PBS containing
0.2% (w/v) Marvel and 0.025% Triton X-100.
ELISA
Microtitre 96-well plates (Nunc; MAXI-Sorp) were coated with anti-human
IgG (Fc-specific) goat antiserum (Sigma-Aldrich) for 1 h at 37˚C. The
plates were blocked (5% Marvel/PBS/0.2% Tween 20) for 1 h at room
temperature; sRgp46-Fc–containing Drosophila medium supernatant was
added and incubated for 1 h at room temperature; and unbound SU was
removed by washing (53). Subsequently, murine envelope-specific and
control Abs in hybridoma supernatants or purified mAb at the concentrations indicated were added and incubated with the immobilized target
Ag for 2 h at room temperature. Plates were washed extensively, and
peroxidase-conjugated anti-mouse IgG (1:10,000 dilution; Sigma-Aldrich)
was added and the bound Ab detected using fresh ABTS substrate at 15
mg/ml (in 0.1 M citric acid, 0.06% H2O2). After 10–20 min, the absorbance was read at 415 nm. Dose-dependent Ab binding was compared by
nonlinear regression using GraphPad by Prism, and minima and maxima
for each data set were constrained to 0 and 100%, respectively.
Syncytium interference assays
Syncytium interference assays were performed by standard methods (28,
29, 43, 44). HeLa cells (2 3 105) transfected with the envelope expression
vector pHTE-1 were added to untransfected HeLa target cells (8 3 105).
The effector and target cells were cocultured in the absence or presence of
the mAbs at the concentrations specified. The cells were incubated for 12–
15 h at 30˚C and returned to 37˚C for 1–2 h, washed twice with PBS, and
fixed in PBS containing 3% paraformaldehyde. Syncytia were stained with
Giemsa’s solution (BDH). Assays were performed in triplicate, and the
number of syncytia from five fields (3200) per replicate was scored by
light microscopy.
Pseudotyping assay
Pseudotyped virus was prepared, as described (26, 45). Briefly, 293T cells
were cotransfected using Genejuice (Novagen) with 10 mg env-defective,
luciferase-encoding, HIV proviral clone pNL4-3.LUC.R-.E- (46, 47) (obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, and donated by N. Landau, New York University School of Medicine) in the presence or absence of 10 mg pHTE-1.
Sodium butyrate (20 mM) was added after 18 h. The sodium butyrate
medium was changed to normal growth medium after 24 h, and the viral
supernatants were harvested after an additional 24-h incubation by centrifugation at 2500 3 g and filtration through a 0.20-mM micropore filter.
For transduction of HeLa or HOS cells, triplicate samples of 7 3 105 target
cells in 1 ml were incubated with 1 ml undiluted or serially diluted virus
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
assayed for luciferase activity following the manufacturer’s instructions
using the Luciferase Assay System (Promega) and a Turner Designs TD20/20 luminometer (Sunnyvale, CA). For Ab inhibition experiments, 1 ml
HTLV-1 Env-pseudotyped virus was incubated with 1 ml HeLa or HOS
cells (4 3 105 cells) in the presence or absence of serial dilutions of purified mAb. After overnight culture, the cells were lysed and assayed for
luciferase activity using the Promega Luciferase Assay System, as directed
by the manufacturer.
Flow cytometry
HTLV-1–infected MT2 and noninfected (control) Sup-T1 cells at a density
of 106 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
1 h with rotation. The cells were pelleted at 1600 rpm for 5 min, in an
Eppendorf C5415C microfuge, washed, and incubated with anti-mouse
FITC-labeled Ab (1:1000 dilution) in RPMI 1640 medium at room temperature for 1 h in the dark. The cells were washed (PBS/0.1% sodium
azide), fixed (0.5% paraformaldehyde in PBS [pH 7.4]), and kept in
the dark at 4˚C until analyzed by flow cytometery (FACScan; BD Biosciences).
Microscopy
MT2 cells (2 3 106 in 2 ml) were incubated with 250 ml SU mAb hybridoma culture supernatant at 37˚C/5% CO2 for 20 min and plated onto
poly-L-lysine–coated glass coverslips (Sigma-Aldrich). The cells were
incubated at 37˚C/5% CO2 on the coverslips for 60 min. Cells were washed
with PBS, fixed with 3% paraformaldehyde for 15 min at room temperature, and then blocked (PBS, containing 10% FBS and 0.25% BSA). The
cell-bearing coverslips were carefully washed three times in blocking
buffer and incubated in the dark with anti-mouse IgG-FITC (1:750; Sigma-
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geometry of native viral envelope and the mode of interaction with
Abs have a profound impact on the ability of Abs to neutralize
virus infectivity. Understanding the immunogenic properties of
HTLV-1 envelope is therefore a critical issue for the development
of effective vaccines and immunological treatments to combat
HTLV-1 infection.
Although the crystal structure for HTLV-1 SU has not yet been
determined, rigorous examination suggests that the SU is comprised of two autonomously folding domains separated by
a proline-rich linker peptide (31–36). The N-terminal region, also
known as the receptor-binding domain (RBD), is necessary and
sufficient for binding to Glut-1 on target T cells (21, 33). Moreover, transfer of the RBD into a heterologous viral envelope
confers upon the recipient virus the cellular tropism that is typical
of HTLV-1 (32, 37). By contrast, the C-terminal domain (CTD) is
less well characterized, but accumulating evidence suggests that
the CTD not only provides an important structural function in the
envelope trimer, but is also involved in recognition of additional
entry factors or coreceptors (38, 39), in determining the differing
tropisms of HTLV-1 and HTLV-2 (40), and in transducing the
envelope activation signal upon receptor binding (23, 24, 41). To
date, the contribution of these domains to recognition by Ab and
to the sensitivity of HTLV-1 to Ab-mediated neutralization is incompletely understood.
In this study, we investigate whether a recombinant form of SU
that is competent for receptor binding can induce Ab responses and
whether such Abs are capable of inhibiting envelope-mediated
membrane fusion and viral entry. We demonstrate that many
anti-SU Abs exhibit some neutralizing activity, whereas some are
potently neutralizing. The most robustly neutralizing Abs tend to
recognize the RBD of SU, but, although the relative binding efficiency of mAbs may be similar, there are marked differences
in the inhibitory properties of RBD-targeted Abs. Moreover, we
demonstrate that even poorly neutralizing Abs have the ability to
recognize HTLV-1 virions and to recruit cytotoxic responses to
HTLV-1–infected cells. Finally, we provide evidence that neutralizing Abs block HTLV-1 infectivity at distinct steps of the viral
entry process. We discuss the implications of our findings for
HTLV-1 immunotherapy.
HTLV-1 SU-SPECIFIC Abs
The Journal of Immunology
Aldrich) at room temperature for 1 h. Coverslips were mounted onto glass
slides with Mowiol (Mowiol 4-88; Calbiochem) mounting medium or with
Vectashield (Vector Laboratories) for differential interference contrast
microscopy. Slides were sealed with nail varnish and left to dry in the
dark before examination by fluorescence microscopy and/or differential interference contrast imaging using a Zeiss Axioplan 2 microscope with
Openlab (Improvision) data acquisition software.
Isolation of peripheral blood polymorphonuclear neutrophils
proteins’ ability to bind in a dose-dependent and saturable manner
to Jurkat and Sup-T1 CD4+ cell lines in flow cytometry assays
(43). The functional sRgp46-Fc was used to immunize CD1 mice,
which responded robustly to the immunogen, and subsequently,
splenocytes were recovered from three mice with high Ab titres
(endpoint dilution .1:20,000). Splenocytes from individual mice
were fused to SP2/0-Ag14 or, alternatively, NSO-1 myeloma cells
and hybridomas selected using standard methods. Over 1700 hybridomas were recovered, of which 168 were positive for activity to the immunogen. From this group of hybridomas, based on
the hybridoma fusion partner, mouse donor, Ab isotype, reactivity
with the recombinant Ag, and failure to interact with human IgG,
18 independent hybridomas reactive with sRgp46-Fc were selected
for detailed study (Fig. 1A). The selection criteria reduced the
likelihood that multiple mAbs were derived from the same clonally
expanded splenocyte. Each of the mAbs bound to sRgp46-Fc, but
did not bind to the control tissue plasminogen activator (tpa)-Fc
fusion product (Fig. 1A), indicating that the binding activity was
directed at the SU component of the envelope-Fc fusion protein.
Isotyping assays indicated that the panel included six IgG1, six
IgG2a, five IgG2b, and one IgM Abs (Table I).
Representative mAbs were purified from conditioned medium,
and the relative binding activities were compared in dosedependent sRgp46-Fc–binding assays (Fig. 1B). Although these
ELISA-based assays do not allow accurate calculation of dissociation constants (Kd) for the Abs, the concentration of Ab required to give half-maximal binding can be used as a comparator
Respiratory burst assay
The Ab-dependent neutrophil respiratory burst was assessed using a
chemiluminescence procedure (48). MT2 cells were plated at 200 ml (2 3
106 cells/ml) per well of a 96-well plate and fixed with 0.05% glutaraldehyde. The cells were centrifuged onto the plate at 1000 rpm (Heraeus;
Labofuge 400) for 10 min, washed with phosphate buffered saline and
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-aminophthalhydrazide [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
analysis of viral protein by Western blotting.
Results
To investigate the immunological properties of HTLV-1 envelope,
a functional soluble form of recombinant SU (rSU), sRgp46-Fc,
which includes amino acid residues Ser25 to Ser308 of envelope
(amino acid coordinates based on the unprocessed envelope primary translation product), was concentrated and purified by Con
A and protein A affinity chromatography (43). The receptorbinding activity of the purified protein was confirmed by the
FIGURE 1. Murine mAbs bind to HTLV-1 SU. A, mAbs derived from
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
coated onto the wells of a 96-well plate; following incubation with conditioned medium supernatant from each of the hybridomas, the plates were
washed and bound Ab was detected. Background reactivity to the control
protein tpa-Fc was subtracted from each of the data points shown. The
negative control represents the signal obtained from the secondary Ab in the
absence of a primary SU-specific murine mAb. B, Dose-dependent binding
of purified mAbs to sRgp46-Fc. mAbs at the concentrations indicated were
incubated with immobilized sRgp46-Fc. Following washing, bound Ab was
detected. All data represent the mean 6 SD of triplicate assays.
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Buffy coat fractions from healthy donors were obtained from the Scottish
Blood Transfusion Service. Polymorphonuclear neutrophils (PMN) were
obtained by standard procedures including dextran sedimentation to remove
erythrocytes and platelets, hypotonic lysis to remove remaining erythrocytes and platelets, and Ficoll sedimentation to separate mononuclear
cells from neutrophils. In brief, buffy coat fractions from donors (50 ml
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/
0.9% NaCl) was then added to the ACD/blood mixture. The ACD/dextran/
blood mixture was incubated at room temperature for 45 min, or until
sedimentation was complete. The separated supernatant was centrifuged at
1000 rpm (Heraeus; Labofuge 400) for 12 min at 4˚C without break. The
cell pellet was resuspended in ice-cold ddH2O, and 0.6 M KCl was added
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
with break, and the process was repeated until no RBCs remained. The
cell suspension was layered onto Ficoll-Hypaque (density 1.077; SigmaAldrich) and centrifuged at 1200 3 g for 30 min at 4˚C, and mononuclear cells were collected at the Ficoll interface, whereas the neutrophils
sedimented to the bottom of the Ficoll, where they were recovered. Purified PMN as effector cells were washed with HBSS, enumerated by
hemocytometry, and resuspended in HBSS/BSA at 2 3 107 cells/ml. Viability was .99%, and PMNs constituted .90% of leukocytes. PMN were
used immediately after harvest and purification.
3
4
HTLV-1 SU-SPECIFIC Abs
Table I. Isotype and affinity of HTLV-1 SU mAb
Hybridoma
Isotype and Subclass
Half-Maximum Binding (mg/ml)
CSK1-9
CSK2-43
CSK2-71
CSK3-13
CSK4-13
CSK4-24
CSK4-34
CSK5-37
CSK7-12
CSK11-5
CSK11-18
CSK11-81
CSK11-83
CSK15-18
CSK16-13
CSK16-47
CSK-N30
CSK-N37
IgG2a
IgG1
IgG1
IgG1
IgG2b
IgG1
IgG1
IgG2a
IgG2b
IgG2a
IgG2b
IgG2a
IgM
IgG2a
IgG2b
IgG2a
IgG2b
IgG1
0.026
ND
0.144
18.40
ND
0.016
0.222
0.037
0.501
0.153
0.075
0.005
ND
0.025
0.315
0.009
0.306
0.275
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.
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of relative binding affinity. Analysis of the binding curves and
calculation by nonlinear regression analysis of the Ab concentration required to give half-maximal binding (Table I) indicated
that there is a ∼50-fold difference in the relative binding activity
within the main group of mAbs. Three Abs, CSK1-9, CSK16-47,
and CSK11-81, exhibit high relative binding to sRgp46-Fc,
whereas mAb CSK3-13 demonstrated the lowest binding activity (Fig. 1B, Table I).
To begin to characterize the regions of SU recognized by SUspecific mAbs, we compared the reactivity of the mAbs to
sRgp46-Fc, two envelope deletion constructs, and a control Fc
protein. These constructs encode SU or its derivatives fused to the
Fc region of human IgG (Fig. 2A) and are reactive with Fc-specific
control antisera. In ELISA, all mAbs bound to sRgp46-Fc, but not
to a control tpa-Fc product (Fig. 2B). Moreover, mAbs CSK2-71,
CSK4-34, CSK11-5, CSK16-13, CSK-N30, and CSK-N37 bound
efficiently to an envelope construct, CTD-Fc, which lacks amino
acid residues 25–190 of SU (Fig. 2). The region deleted in these
constructs encodes all of the amino acids currently known to be
important for binding to the receptor Glut-1 (32). Therefore,
CSK2-71, CSK4-34, CSK11-5, CSK16-13, CSK-N30, and CSKN37 recognize epitopes that lie in the CTD of SU, in a region that
is not required for interaction with Glut-1. By contrast, most of the
remaining mAbs bound only to envelope proteins that included the
N-terminal receptor-binding domain (RBD-Fc) of SU (residues
Ser25 to Leu190), but did not bind to envelope fragments that lack
these sequences (Fig. 2), indicating that the majority of the mAbs
recognize the RBD of SU. Only CSK2-43 bound to both the RBDFc and CTD-Fc proteins, but did not bind to tpa-Fc, indicating that
this Ab most likely recognizes an epitope that overlaps the junction of the RBD and CTD (Fig. 2B). In addition, each mAb was
examined for recognition of denatured Ag by Western blot analysis against sRgp46-Fc, rSU (without the Fc-tag), and envelopederived fragments (Fig. 2C). Although raised against intact functional Ag, all of the mAbs displayed efficient binding to denatured envelope, including the untagged SU. Therefore, the Abs
The Journal of Immunology
are not dependent on the conformation of the Ag for efficient
binding and most likely recognize linear epitopes of SU. The
Western analysis also confirmed the domain specificity of each
mAb.
Candidate immunogens for subunit vaccines must routinely and
robustly elicit Abs that recognize the native, extensively glycosylated, and trimeric structures of viral envelope. To explore the
reactivity of anti-SU mAbs with virally expressed envelope, several
complementary assays were employed. First, binding of each mAb
to HTLV-1–infected MT2 cells was examined by flow cytometry. The majority of the mAbs bound efficiently to the HTLV-1–
infected cells (Fig. 3A, 3B), whereas none of the mAbs bound to
the control Sup-T1 cells. These results indicate that the majority
of the mAbs raised against sRgp46-Fc recognize viral envelope
displayed on the surface of infected human T cells. By contrast,
several mAbs, CSK2-43, CSK3-13, and CSK11-83, bound poorly
to HTLV-1–infected cells (Fig. 3B), but bound effectively to
sRgp46-Fc (Figs. 1, 2) and recognize SU by Western blotting (data
not shown). The simplest and, in our view, most likely explanation
5
Ab-dependent neutralization of envelope-mediated membrane
fusion and viral entry
FIGURE 3. Murine mAbs recognize HTLV-1 envelope displayed on
infected cells. A, Chronically infected MT2 cells were incubated with anti-SU
mAbs, or irrelevant control mAb (anti-Fc), as indicated (open histograms), or
control MT2 cells probed in the absence of primary Ab (solid histogram).
Bound Ab was detected using FITC-conjugated anti-murine IgG antisera and
flow cytometry. Typical results are illustrated for Abs CSK1-9, CSK3-13,
CSK4-24, and CSK11-81. No binding was observed with uninfected Sup-T1
cells (data are shown in Supplemental Fig. 1). B, All mAbs were examined
for the ability to bind to MT2 cells, as described above, and the results displayed as mean fluorescence intensity of the cell population after subtraction
of the background signal produced in the presence of secondary Ab alone
(typical data from one of multiple independent assays [n = 3] are shown). C,
Typical immunofluorescence images (original magnification 3630) of HTLV1–infected MT2 cells probed with the anti-SU mAbs CSK1-9, CSK4-24,
CSK11-5, and CSK16-47. These images are typical of those obtained with all
MT2 immunofluorescence-positive mAbs (Table II). No binding was observed with uninfected Sup-T1 cells by microscopy or by flow cytometry (see
Supplemental Fig. 1).
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
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. Surprisingly, 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
various Ab classes and RBD or CTD specificities was selected for
high-yield purification and examined for the ability to inhibit
membrane fusion in syncytium interference assays (Fig. 5A). HeLa
cells transfected with the subgenomic HTLV-1 envelope expression construct pHTE-1 were cocultured with nontransfected HeLa
target cells in the presence of purified mAb. In the absence of Ab,
or in the presence of irrelevant anti–HIV-1 gp120 control mAb
(178.1), extensive membrane fusion and syncytia production were
observed (Fig. 5A, 5B). By contrast, several of the anti-SU mAbs,
for example CSK7-12, CSK11-18, CSK4-24, and CSK1-9, efficiently blocked syncytium formation, as observed by low-power
microscopy (Fig. 5A, 5B). Although CSK7-12 has modest relative
binding activity to gp46 (Fig. 1B, Table I), mAbs CSK11-18,
CSK4-24, and CSK1-9 exhibit strong relative binding to rSU,
and all of these mAbs efficiently recognize viral envelope in flow
cytometry assays. In contrast, some mAbs, CSK4-34, CSK15-18,
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for these results is that the epitopes recognized by these Abs are
masked or occluded on the intact viral envelope complex and that
removal of SU from TM, or denaturation of SU, increases the
exposure of these epitopes. However, Env has been observed to
interact with neuropilin 1 and Glut-1 in HTLV-1–infected cells
(21, 22); consequently, an alternative scenario is that such interactions may prevent binding of these mAbs to envelope on
infected cells. Although useful in Western analysis, due to their
lack of reactivity with native viral envelope, mAbs CSK2-43,
CSK3-13, and CSK11-83 were not studied further. Generally,
mAbs that displayed strong relative binding to sRgp46-Fc also
exhibit robust binding to infected T cells.
To visually confirm binding of mAbs to viral envelope, chronically infected MT2 cells were probed with SU-specific mAbs and
examined by fluorescence microscopy. The majority of mAbs that
were reactive with envelope in flow cytometry studies also demonstrated binding to viral envelope by fluorescence imaging.
Typically, mAbs exhibit homogenous staining around the plasma
membrane of unstimulated MT2 cells (Fig. 3C) consistent with the
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.
6
and CSK16-47, antagonized membrane fusion weakly even at
high mAb concentrations. Notably, although CSK16-47 binds to
the RBD of SU, is among the Abs with highest relative binding
activity to monomeric SU, and very efficiently binds to virally
infected cells, it is a relatively weak inhibitor of syncytium formation even at high Ab concentrations (Fig. 5A). By comparison,
the most potently neutralizing Abs within this panel, CSK7-12,
CSK11-18, CSK4-24, and CSK1-9, also recognize the RBD of
SU. Therefore, mAbs CSK7-12, CSK11-18, CSK4-24, and CSK19 were compared directly for inhibition of membrane fusion (Fig.
5C). In each case, the Abs displayed potent dose-dependent inhibition 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,
a mAb with high relative binding affinity, was less effective than
CSK7-12 (Fig. 5). Therefore, the ability to inhibit membrane fusion does not correlate strictly either with the efficiency of binding
to envelope or recognition of the RBD.
To examine the ability of the mAbs to block envelope-mediated
entry of free viral particles, envelope-deficient HIV-based luciferase-transducing viral particles were pseudotyped with HTLV-1
envelope and used in interference of infectivity assays. Each of
the mAbs that efficiently blocked syncytium formation also
FIGURE 5. Many SU-specific mAbs display neutralizing activity. The
ability of murine mAbs to inhibit Env-dependent membrane fusion was
examined in syncytium interference assays. A, HeLa target cells were cocultured 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 percentage of inhibition of syncytium formation relative to untreated controls. B,
Typical Giemsa-stained monolayers for control cells in the absence of envelope 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
the irrelevant control Ab 178.1 (HIV-1 gp120 specific) are illustrated. C, The
most robustly neutralizing Abs CSK1-9, CSK4-24, CSK7-12, and CSK11-18
were compared in dose-dependent syncytium interference assays. Briefly,
target and effector cells were mixed in the presence of increasing doses of
mAb, and the percentage of inhibition of syncytium formation was determined. All assays were performed in triplicate, and the number of syncytia
from five fields (original magnification 3200) per replicate was scored by
light microscopy.
blocked entry of HTLV-1 envelope-pseudotyped viral particles
(HTLV-1pp). Robust transduction of luciferase was observed in the
absence of Ab or, alternatively, in the presence of increasing doses
of an irrelevant control Ab. By contrast, a strong and dosedependent inhibition of HTLV-1pp entry and luciferase transduction was observed for mAbs CSK7-12, CSK4-24, and CSK1-9
(Fig. 6). Again, inhibition of HTLV-1pp entry did not correlate
strictly with Ab affinity; although CSK1-9 is one of the strongest
binding Abs, it did not inhibit as potently as CSK7-12, especially
at low Ab concentrations.
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FIGURE 4. Some mAbs efficiently block binding of sRgp46-Fc to human 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 antiSU mAb; open green histogram, Sup-T1 cells in the presence of sRgp46Fc and the indicated SU-specific murine mAb. In the top left control panel,
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
the method described above. Data are plotted as percentage of inhibition of
maximal fluorescence relative to cells probed in the absence of SU-specific
mAbs. All data values represent the mean of triplicate samples.
HTLV-1 SU-SPECIFIC Abs
The Journal of Immunology
FIGURE 6. Anti-SU–specific mAbs neutralize viral infectivity. Murine
mAbs (as indicated) were incubated with uninfected Sup-T1 cells in the
presence of HIV-1 luciferase-transducing viral particles pseudotyped with
HTLV-1 envelope. Following infection and recovery, the cells were assayed for transduced luciferase activity and the data were plotted as percentage of inhibition relative to untreated viral particles.
It was intriguing that despite recognizing SU and interacting with
envelope on the surface of infected cells, some mAbs are unable to
inhibit membrane fusion or neutralize the entry of HTLV-1pp. A
plausible explanation for the lack of neutralizing activity of some
SU-reactive mAbs is that, contrary to neutralizing mAbs, nonneutralizing mAbs do not bind effectively to trimeric envelope
displayed on the surface of cells or virions. We therefore tested
the ability of two neutralizing mAbs (CSK1-9 and CSK11-81),
one weakly neutralizing (CSK16-47), and one nonneutralizing
(CSK16-13) mAb of similar relative binding affinity to immunoprecipitate HTLV-1 particles produced by chronically infected
MT2 cells. Surprisingly, both neutralizing and poorly neutralizing
mAbs were effective at capturing virus particles (Fig. 7), indicating
that the poor neutralizing activity is not associated with a failure to
recognize viral envelope on the virion surface.
Ab-dependent recruit and activation of immune effector cells to
HTLV-infected cells
nescence-based in vitro assay (48). Samples of HTLV-1–infected
MT2 cells were opsonized with each of the independent SUspecific 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
HTLV-1–infected T cells. By contrast, MT2 cells opsonized with
anti-SU mAbs markedly enhanced the PMN respiratory burst (Fig.
8) and, significantly, both robustly and weakly neutralizing Abs
induced strong responses in these assays. In particular, CSK11-18
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 neutralizing (Table II). Thus, the ability to recruit and activate immune
effector cells is not tightly coupled to the neutralizing proficiency
of HTLV-specific Abs.
Discussion
HTLV-1 infections induce adaptive immune responses to viral Ag,
but, despite robust immune activation, infected individuals fail to
clear virus and virally infected cells. The accumulating data suggest
that there is an ongoing war of attrition between the immune system
and the virus, wherein the immune response acts to eradicate the
virus and the virus employs diverse, but inadequately understood
mechanisms to avoid immune surveillance. A clear view of the
immunological pathways and viral targets required for successful
suppression or eradication of HTLV-1 replication is critical to the
development of vaccines and novel immunological strategies to
combat HTLV-1–associated disease. As a step toward this objective, we in this study demonstrate that a recombinant HTLV-1
envelope-derived immunogen displays properties of value to subunit 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 7. Murine mAbs with markedly differing neutralizing activities recognize envelope displayed on HTLV-1 virions. Virions from cellfree MT2 supernatants were immunoprecipitated with the anti-SU monoclonals CSK1-9, 11-81, 16-13, and 16-47, and the irrelevant HIV-1 gp120specific control Ab 178.1, or in the absence of Ab. Captured viral particles
were processed and analyzed for genomic RNA by RT-PCR and gel
electrophoresis (top panel; control = template-free RT-PCR reaction), or
for viral capsid protein P19 content by Western blotting (lower panel).
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
isolated human neutrophils. The PMN respiratory burst was assayed by
standard procedures using a luminol turnover method (48). The respiratory
burst of neutrophils, shown as maximal relative light units, in the presence
of opsonized MT2 cells, was compared with neutrophils incubated in the
presence of MT2 cells opsonized by an irrelevant anti-murine Fc mAb,
anti-Fc; or with MT2 cells in the absence of Ab, PMN + MT2; or neutrophils in the presence of uninfected Sup-T1 cells, PMN + Sup-T1; or
unstimulated neutrophils, PMN.
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Both neutralizing and nonneutralizing mAbs bind to viral
particles
7
8
HTLV-1 SU-SPECIFIC Abs
Table II. Summary of mAb properties
mAb
Isotype
Region
Immunofluorescence
Inhibition of
SU Binding
Inhibition of
Syncytium Formation
Inhibition of
Virus Entry
CSK1-9
CSK2-43
CSK2-71
CSK3-13
CSK4-13
CSK4-24
CSK4-34
CSK5-37
CSK7-12
CSK11-5
CSK11-18
CSK11-81
CSK11-83
CSK15-18
CSK16-13
CSK16-47
CSK-N30
CSK-N37
IgG2a
IgG1
IgG1
IgG1
IgG2b
IgG1
IgG1
IgG2a
IgG2b
IgG2a
IgG2b
IgG2a
IgM
IgG2a
IgG2b
IgG2a
IgG1
IgG2b
RBD
CTD
CTD
RBD
RBD
RBD
CTD
RBD
RBD
CTD
RBD
RBD
RBD
RBD
CTD
RBD
CTD
CTD
+++
+
+
2
2
+++
+++
2
+++
+++
++
+++
ND
2
+++
+++
+++
+++
++++
2
2
2
+
++
++
2
++++
2
++++
+++
2
2
2
+++
++
++
+++
ND
ND
ND
ND
+++
+
ND
++++
ND
+++
++
ND
2
2
+
+
ND
+++
ND
ND
ND
ND
+++
2
ND
+++
ND
+++
ND
ND
ND
ND
ND
2
ND
have enabled the selection of a diverse panel of SU-specific mAbs
that will facilitate analysis of the immunological features of envelope.
Significantly, the murine mAbs exhibit properties that are typical
of Abs produced during natural infections. The mAbs bind to
virally expressed envelope displayed both on infected cells and,
most importantly, on virion surfaces. The majority of the mAbs
possess some neutralizing activity, and a few are robustly neutralizing in assays of membrane fusion and pseudovirus entry. The
neutralizing activity of mAbs in syncytium interference assays is
particularly pertinent to natural infections because much of the
viral dissemination in infected individuals occurs by transfer of
virus between cells across regions of close cell-to-cell contact (50,
51). Syncytium formation may therefore be viewed as an extreme
outcome of envelope-mediated processes that naturally occur
within regions of close membrane apposition. Notably, the most
effective neutralizing activity is associated with Abs that recognize the RBD of SU. Nevertheless, despite similar relative binding
efficiencies, there are marked differences in the neutralizing proficiencies of RBD-specific mAbs, indicating that not only affinity,
but also epitope recognition determines the neutralizing properties
of anti–HTLV-1 Abs. Furthermore, some poorly neutralizing Abs
exhibit efficient recognition of HTLV-1–infected cells; perhaps,
such Abs recognize alternative forms of envelope that are not
compatible with membrane fusion and viral entry (discussed in
detail below).
The neutralizing properties of the HTLV-1 SU-specific mAbs
contrast strikingly with the activity of mAbs previously raised
against the fusion-associated structures of TM (29, 30, 52). Despite
the ability of anti-TM mAbs to block binding of leash and helical
region-derived peptides to the coiled coil in vitro, to recognize cell
surface displayed viral envelope, and to target a region critical to
the membrane fusion process, these Abs unanimously failed to
inhibit either envelope-mediated membrane fusion or viral entry
into cells (29, 30, 52). By contrast, most of the mAbs targeted to
SU exhibit some level of neutralizing activity. The collated data
suggest that in the fusion-active envelope structure, the coiled coil
of TM is poorly accessible to Ab and is therefore protected from
the neutralizing activity of TM-targeted mAbs. Conversely, SU
appears to be sensitive to neutralization throughout the receptorbinding and entry process. In particular, our data demonstrate
that some mAbs (CSK1-9, CSK7-12, CSK11-18) effectively
block binding of the SU to cell surface receptors, whereas others
(CSK15-18) block fusion at a postreceptor-binding step of entry
(Figs. 4, 5). Interestingly, based on the observation that the neutralizing proficiency exceeds the ability of the Ab to prevent SU
binding to receptor, we find that mAbs such as CSK4-24 appear to
interfere with both receptor binding and the postbinding events of
entry. To our knowledge, this is the first data to successfully resolve the ability of Abs to neutralize HTLV-1 entry by disrupting
independent steps of the binding, fusion, and entry process.
Alkylation studies coupled with elegant cryo-electron microscopy (23, 24, 41, 53) demonstrate that, upon receptor binding, the
envelope protein of MLV undergoes a radical change in conformation. Receptor binding induces an isomerization and disruption
of the SU-TM intersubunit thiol, which triggers the reorganization
of the SU subunits to form an open ring-shaped structure. It has
been suggested that the TM subunit may extend through the center
of the trimeric SU complex to induce membrane fusion (41). If
this is indeed the case, the critical fusion-active surfaces of TM are
likely to be sterically shielded from neutralizing Ab due to the
presence of the SU subunits and the proximity of the viral and cell
membranes. By comparison, SU subunits are likely to have surfaces exposed to Ab in the prefusogenic state and following
binding of SU to cell surface receptors. Our observations that
some SU-specific Abs block the postreceptor-binding steps of
entry indicate that such mAbs may interfere with coreceptor or
cofactor recruitment (38, 39) or, alternatively, may prevent the
receptor-induced changes in SU conformation that are required to
activate the fusogenic properties of envelope. Understanding the
molecular mechanism by which these mAbs neutralize viral entry
will provide greater insight into envelope function and the immunology of HTLV-1 infections.
A perplexing aspect of these studies is that despite efficient
recognition of the SU on infected cells and the ability to bind to
envelope on viral particles, some mAbs are poor inhibitors of
membrane fusion and viral entry. It is difficult to reconcile binding
of a large Ab (150 kDa) to a small surface glycoprotein (46 kDa)
without loss of protein function. Indeed, for HIV-1, a compelling argument has been made that if an Ab binds to the wildtype envelope complex, it will neutralize (54–57). Consistent with
this notion, a heterologous Ab can inhibit and neutralize HIV-1
infectivity when the epitope recognized by that Ab is engineered
into the gp120 subunit of HIV-1 envelope (56, 57). By contrast,
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++++, very strong activity; +++, strong activity; ++, moderate activity; +, weak activity; 2, no activity; ND, not determined.
The Journal of Immunology
HIV-1, pose unique technical challenges and have often suffered
disappointing setbacks. Nevertheless, HTLV-1 is an attractive target
for vaccine development. The HTLV-1 envelope glycoprotein shows
relatively little sequence variation in and between infected individuals
(65); envelope is a primary target for humoral and cell-based immune
responses in natural infections; an attenuated strain of the related
bovine leukemia virus induces sustained resistance to experimental
bovine leukemia virus challenge (18); an envelope-based HTLV-1
immunogen provides protection in primate models of HTLV-1 infection (16, 17); and finally, infants born to HTLV-1–infected mothers
are protected from HTLV-1 infection in the first months of life by
maternally acquired Abs (15). Collectively, these observations suggest that an effective envelope-based subunit vaccine is an achievable
objective for therapeutic intervention in HTLV-1 infections.
There are significant challenges to address before an effective
HTLV-1 vaccine can be realized. For example, the native prefusogenic structures and fusion-active forms of envelope may both
be targeted by Ab, but it is not clear which of these forms is most
appropriate to vaccine design. Moreover, it is probable that a trimeric form of envelope would produce a more effective, longerlasting response than a monomeric subunit approach, but this
awaits experimental validation. Our study provides information and
insight into the way that HTLV-1 envelope interacts with the humoral immune response and how these interactions may be important for viral pathogenesis and ultimately for future vaccine
initiatives. A successful vaccine strategy for HTLV-1 would have
considerable clinical impact and could act as a useful pathfinder for
the more intractable and highly mutable viruses such as HIV-1.
Acknowledgments
We thank Lisa Orram for assistance with the purification of Abs, Dr. Kulpash
Nurkiyanova for technical assistance with hybridoma fusion, Dr. Abdenour
Soufi for independent confirmation of results, Dr. Daniel Lamb and members of the laboratory for useful discussions, and the East Scotland Blood
Transfusion Services for supplying leukopacks. We also thank Dr. Jenny
Woof and Dr. Lindsay Tulloch for helpful comments on the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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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.
A significant finding of our study is that both neutralizing and
weakly neutralizing Abs recruit and robustly activate the cytotoxic
responses of neutrophils. A recent study also demonstrated that
nonneutralizing Abs are capable of recruiting complement to envelope and envelope-expressing cells (29). Our observations indicate 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
cellular cytotoxicity may be of particular benefit in suppression of
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
as the virological synapse, rather than through infection by free
viral particles (50). Due to the close contact of the infected cell
and uninfected cell membranes during cell-to-cell transmission,
HTLV-1 may be protected from the neutralizing properties of Ab,
but, because of cell surface display of envelope, infected cells
should remain sensitive and vulnerable to Ab-dependent cellular
cytotoxicity and Ab-mediated complement fixation. Nonetheless,
despite compelling evidence that effector function has a profound
impact on the success of passive immunotherapy for HIV-1, for
other viral infections, and for treatment of solid tumors (60, 61),
the role of Ab effector function has been largely neglected in
studies of immune control and pathogenesis of HTLV-1 and other
viral infections (61). Although clearly important, neutralizing proficiency is only one aspect of Ab function that should be considered in the design of effective immunotherapies to control viral
load and HTLV-1–associated disease.
Vaccine strategies remain the primary means of combating viral
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