Conformation-specific antibodies targeting the trimer-of-hairpins motif of the human T-cell leukemia virus type 1 transmembrane glycoprotein recognize the viral envelope but fail to neutralize viral entry

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections.

FIG. 1.

HTLV-1 TM and the recombinant TM fusion proteins. (a) Structure of the trimer-of-hairpins motif of HTLV-1 TM. The central triple-stranded coiled coil is shown in the space-filling form, with the extended antiparallel peptide and the C-helical region shown in color. (b) Model of the TM-derived MBP-Hairpin immunogen used in this study. The structure is displayed with each monomer of TM in ribbon format fused to maltose binding protein (MBP; white space-filling model). (c) Representation of the functional regions of HTLV-1 TM; the N-helical and C-helical regions are indicated as boxes; amino acid coordinates based on the envelope protein precursor are shown. The glycosylation site in viral TM is indicated by the branched structure. Boxed regions below TM highlight the motifs that are fused to MBP. All structures were modeled using MacPymol software using the coordinates provided by Kobe et al. (44), Protein Data Bank, Brookhaven National Laboratory, PDB ID 1MG1.

FIG. 2.

Murine monoclonal antibodies bind to the trimer-of-hairpins form. Monoclonal antibodies derived from immunized mice were examined for reactivity to MBP-Hairpin and control MBP. The antigens (MBP and MBP-Hairpin) were coated onto the wells of a 96-well plate and, following incubation with conditioned medium supernatant from each of the hybridomas, bound antibody was detected. The data represent the means and standard deviations of triplicate assays.

FIG. 3.

Dose-dependent binding of MAbs to the trimer-of-hairpins structure. Purified MAbs at the concentrations indicated were incubated with immobilized MBP-Hairpin. Following washing, bound antibody was detected. The data represent the means and standard deviations of triplicate assays.

FIG. 4.

Epitopes recognized by anti-TM MAbs are lost on denatured antigen. Each of the purified monoclonal antibodies at the concentrations indicated was examined for the ability to bind to native (closed squares) or denatured (closed triangles) MBP-Hairpin. Native or denatured (boiled in SDS and DTT and diluted in the presence of a carrier protein) antigen was captured on the solid phase by using polyclonal anti-MBP rabbit sera; subsequently, the anti-TM MAbs were each incubated with the immobilized antigen and following extensive washing, bound MAb was detected. As a control for antigen capture, the native and denatured antigens were also examined for reactivity with the monoclonal antibody M20-1, which binds to a linear epitope of MBP (top left graph). The data represent the means and standard deviations of triplicate assays.

FIG. 5.

Oligomerization states of the MBP-TM-derived chimeras. Gel filtration chromatography profiles of the trimeric MBP-hairpin (T^H), BSA, and monomeric MBP control proteins are indicated (top trace). The profiles of the MBP-N-helix, MBP-C-helix, and MBP-CX6CC-loop fusion proteins are shown below. The predicted molecular masses of each protein monomer are as follows: MBP, 43 kDa (lane 2); MBP-Hairpin, 53 kDa (lane 3); MBP-N-helix, 48 kDa; MBP-C-helix, 46 kDa; and MBP-CX6CC-loop, 46 kDa. A small amount of aggregated material can be observed in the profile for MBP-CX₆CC-loop (approximate elution volume, 10 to 13 ml); this is due to nonspecific intermonomer disulfide bonding and can be reduced in the presence of 5 mM DTT. Calibration markers (arrows above plots) are ferritin, 440 kDa; aldolase, 158 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; and RNase A, 13.7 kDa.

FIG. 6.

Murine MAbs bind to both the trimer-of-hairpins and coiled-coil structures of TM. Purified antibody (a) or antibody in hybridoma-conditioned medium supernatant (b) was examined for binding to a panel of recombinant TM derivatives (shown in Fig. 1). The antigens MBP, MBP-Hairpin, MBP-Fishhook, MBP-N-helix, MBP-C-helix, and MBP-CX₆CC-loop were each adsorbed onto the solid phase. Purified antibody (1 μg/ml) or conditioned medium supernatants (50 μl) were incubated for 1 h with each of the immobilized antigens, immobilized complexes were washed, and the bound antibody was detected. The data represent the means and standard deviations of triplicate assays and are typical of multiple independent experiments.

FIG. 7.

Murine MAbs recognize envelope expressed on virally infected cells. HTLV-1-infected cells (MT2) were incubated with irrelevant primary anti-MBP (M20-1) or the anti-TM MAbs as indicated. Bound antibody was detected using FITC-conjugated anti-mouse antibody and flow cytometry. The solid histograms represent the basal fluorescence of the cell population in the absence of primary antibody, and open histograms represent the fluorescence signal in the presence of the specified primary antibody. As negative controls, binding of the antibodies to uninfected SupT-1 cells was also examined. With SupT-1 cells, no increase in fluorescence of the cell population was observed with any of the test or control antibodies (data not shown). The data are typical of multiple independent experiments.

FIG. 8.

Cell surface expression of epitopes recognized by anti-Hairpin MAbs. HTLV-1-infected (MT2) and uninfected control cells (SupT-1) were incubated with anti-Hairpin MAbs, and bound antibody was detected using FITC-conjugated anti-mouse secondary (upper panels); corresponding phase-contrast images are also shown (lower panels). A typical result for monoclonal antibody M8-24 is shown, demonstrating peripheral staining of the plasma membrane on infected cells but not uninfected cells. All antibodies demonstrating positive staining by flow cytometry analysis also exhibited staining patterns essentially identical to M8-24 by fluorescence microscopy (data not shown).

FIG. 9.

Monoclonal antibodies raised against recombinant TM recruit complement. Immobilized MBP-Hairpin was incubated (1 h) with (a) each of the purified anti-Hairpin antibodies or with (b) hybridoma-conditioned medium supernatants (M17-21p, purified M17-21 control; M17-21s, conditioned medium supernatant control). As controls, antigen was also incubated in the absence of antibody or in the presence of irrelevant antibody (anti-HIV gp120 MAb 178.1 or anti-MBP MAb M20-1 or the irrelevant MAb K18-8). Unbound antibody was washed away, and whole serum (as a source of complement) was added and incubated with the Hairpin-antibody complexes. Subsequently, unbound complement was washed away, and bound C1q was detected. The results represent the means and standard deviations of triplicate assays.