JOURNAL OF VIROLOGY, Aug. 1992, p. 4784-4793
Vol. 66, No. 8
0022-538X192/084784-10$02.00/0 Copyright © 1992, American Society for Microbiology
A Monoclonal Antibody to CD4 Domain 2 Blocks Soluble CD4-Induced Conformational Changes in the Envelope Glycoproteins of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-1 Infection of CD4+ Cells JOHN P. MOORE,l,2* QUENTIN J. SATTENTAU,3 P. J. KLASSE,2 AND LINDA C. BURKLY4
Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom2; The Aaron Diamond AIDS Research Center, 455 First Avenue, New York, New York 100161*; Centre d'Immunologie de Marseille-Luminy, 13288 Marseille Cede-x 9, France3; and Biogen Inc., Cambridge, Massachusetts 021424 Received 19 March 1992/Accepted 6 May 1992
The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp4l and dissociation of gpl20 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gpl20/gp41 and/or CD4 that are necessary for virus-cell fusion.
pH-independent fusion of human immunodeficiency virus
proteinase (38); exposure of gp4l epitopes cryptic prior to sCD4 binding (38); and complete dissociation of gpl20 from gp4l (15, 18, 34, 36). Whether any or all of these changes are relevant to the virus-cell fusion process has been a matter for debate (1, 31, 35). In this study, we have tested the effect of anti-CD4 monoclonal antibodies (MAbs) on the conformational changes outlined above. One of these MAbs, 5A8, which binds to D2, potently inhibits HIV-1 infection without blocking gpl20 binding to CD4+ cells (4). Here we report evidence that 5A8 blocks sCD4-induced conformational changes in the HIV envelope glycoproteins. We suggest that 5A8, and other MAbs of this type (6, 16, 17), might block HIV infection by inhibiting similar reactions necessary for viruscell fusion. Our observations might also be relevant to the mechanism of action of protective polyclonal antibodies to simian CD4 raised in rhesus monkeys immunized with human sCD4 (42).
type 1 (HIV-1) with the plasma membrane of CD4+ cells is initiated by the binding of virions to domain 1 (D1) of CD4
(10, 20, 22, 26-28, 39; for a review, see reference 31). The mechanism of the fusion reaction is not known in any detail for HIV-1, or indeed for any retrovirus, but we and others have suggested that the binding of the HIV-1 surface (SU) glycoprotein gpl20 to CD4 induces conformational changes in the envelope glycoproteins and in CD4 (1, 2, 6, 17, 21, 35, 38). These molecular rearrangements are presumed to be necessary to establish an interaction of the fusion domain at the amino terminus of the transmembrane (TM) glycoprotein gp4l with the target cell membrane. Accessory cell surface factors seem likely to be involved at some stage of the fusion process, but their nature and role are at present obscure (3, 7, 25). Binding of HIV-1 virions to CD4+ cells can be detected biochemically (27, 28) and by electron microscopy (14), and fusing virions can be observed by the latter technique (14). HIV-1 cell fusion has also been monitored by a dye redistribution assay (11). However, there are at present no methods available to study conformational changes occurring during the interaction of virions with cell surface CD4. To gain some insights into the fusion reaction, we have therefore studied the interaction of soluble CD4 (sCD4) with HIV-1 virions and the surface of HIV-1-infected cells. With these model systems, we can obtain evidence that suggests the occurrence of conformational changes. This evidence includes temperature-dependent increases in the binding of sCD4 to virion-bound gpl20, which is consistent with a complex binding reaction (12, 32, 34, 40); sCD4-induced increases in the binding of antibodies to the V3 region of gp120 and its cleavage by an exogenous *
MATERIALS AND METHODS Antibodies and sCD4. MAbs 6H10 (D1), 5A8 (D2), F91-55 (D2), OKT4 (D3), and L120 (D4), mapping to the CD4 domains listed in parentheses, have all been described elsewhere (4, 6, 17). MAb 5A8 recognizes an epitope in CD4 D2 with the critical residues being between 121 and 134, as defined by chimeras of mouse and human CD4 and CD4 substitution mutants (4). The human anti-gp4l MAbs 50-69 and 98-6 were kind gifts from S. Zolla-Pazner. MAb 50-69 recognizes a peptide with residues 579 to 613, provided that Cys-598 and -604 are disulfide linked (43). MAb 98-6 competes for binding to gp4l with a MAb that recognizes a peptide with the sequence 644 to 663 (43). All MAbs, and Fab fragments of 5A8 made by conventional techniques (4), were used as purified immunoglobulins. Streptavidin-phyco-
Corresponding author. 4784
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erythrin and anti-mouse phycoerythrin were purchased from Immunotech S.A. (Marseille, France). sCD4 produced as previously described (13) was from Biogen Inc. (Cambridge, Mass.). V'iruses. Virus-containing supernatants of the cell cultureadapted HIV-1 isolates HTLV-III RF (RF) and HTLV-IIIB (IIIB) were obtained by cocultivation of infected and uninfected H9 cells as previously described (29). Virus titers for the stocks used in these experiments were typically 106 to 107 50% tissue culture infective doses (TCID50) per ml. Aliquots were stored under liquid nitrogen and each was used once only. Infectivity assays. The ability of CD4 MAbs to inhibit infection of C8166 cells by RF was assessed by incubating the MAbs (in triplicate) at a range of concentrations (1 to 500 nM, or 9 to 1,200 nM for 5A8 Fab) with 2 x 104 cells in 100 ,ul of RPMI 1640 medium-10% fetal calf serum for 20 min at room temperature. RF (12.5 to 125,000 TCID50 per 50 ,ul) was then added and the plates were incubated at 37°C in the presence of 5% CO2. After 3 days, aliquots (50 ,ul) of supernatants were collected, inactivated with 1% Empigen detergent, and analyzed for p24 content (36). After 7 days, syncytia were scored by light microscopy. The median MAb concentrations giving complete inhibition of syncytium formation or 99% inhibition) were recorded. To distinguish between inhibition of the initial infection by cell-free virus in the inoculum and inhibition of subsequent viral spread by cell-to-cell fusion or infection by progeny virus, the above assay was compared with one in which the cells were first incubated with the virus for 2 h at 37°C and then the MAbs were added. A weaker effect of the antibodies in the latter than in the former assay would be evidence of inhibition of initial infection. Syncytium inhibition assay. H9 cells were infected with RF at a multiplicity of infection of approximately 0.2. After 3 to 4 days of culture, they were washed once in RPMI 1640 medium, resuspended at 2 x 105 cells per ml, and diluted serially in eight twofold steps. 5A8 or 6H10 was diluted in twofold steps and incubated at final concentrations from 67 nM with 4 x 105 C8166 cells per ml in 96-well culture plates (100 p.1 per well) for 20 min at room temperature. A range of dilutions of RF-infected H9 cells (50 ,u1 per well) was then added, and the cocultures were incubated for 24 h at 37°C. The wells were examined by light microscopy for the presence of syncytia. gpl2O-sCD4 enzyme-linked immunosorbent assay (ELISA). RF virus in RPMI 1640 medium containing 10% fetal calf serum was disrupted with 1% Nonidet P-40 (NP-40) (RPMINP-40) to provide a source of soluble gpl20. This was incubated at 50 pM gpl20 per sample with preformed MAbsCD4 complexes in a total volume of 100 p.1 for 1 h at 37°C. The mixtures were supplemented with 2% nonfat milk powder (Marvel; Cadbury Ltd.) and gpl2O-sCD4 complexes (+ bound MAb) were then captured onto a solid phase by adsorbed antibody D7324 to the carboxy terminus of gpl20
(30). Bound sCD4 was detected with a cocktail of MAbs 5A8, L120, and OKT4, each at a saturating concentration of 0.3 ,ug/ml, followed by alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulin G (IgG) (RaMIg-AP). Datum points are the means of triplicate determinations, with standard deviations typically within 10% of the means if not shown. sCD4 binding to virions and gpl20 dissociation. sCD4 binding to intact virions was assessed by a combination of
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gel filtration and ELISA (34, 36). Briefly, virions incubated with sCD4 and/or MAbs were separated from unbound sCD4 and MAbs by gel filtration on Sephacryl S-1000 and then disrupted with 1% NP-40. gpl20 was captured onto a solid phase by antibody D7324. Bound gpl20 was then detected with a pool of HIV+ human sera and alkaline phosphataseconjugated goat anti-human Ig. Alternatively, sCD4-gpl2O complexes were detected with an appropriate pool of antiCD4 MAbs and RaMIg-AP. In some experiments, virionbound MAb was detected with RaMIg-AP after capture of gpl20 onto the solid phase as described above. Note that in this assay, signals can be obtained only if a ternary complex of MAb-sCD4-gpl2O forms on the virion prior to gel filtration. Proteolytic cleavage of the gp120 V3 loop. Thrombin cleavage of gpl20 into 70- and 50-kDa fragments was measured by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) as described previously (38). Exposure of gp4l epitopes. sCD4-induced epitope modulation was measured by a previously described method (38), with the following modifications. (i) Biotinylation of sCD4. In order to detect sCD4 precomplexed with CD4 MAb and bound to the surface of infected cells, sCD4 was biotinylated by the following procedure. Twenty microliters of a solution of sCD4 (130 p.M) in Ca2+and Mg2+-free phosphate-buffered saline (PBS), was diluted sixfold in 100 mM sodium hydrogen carbonate buffer at pH 8.5 and rapidly mixed with 80 p.g of NHS-LC-biotin (Pierce, Rockford, Ill.) per ml. After 24 h of incubation at 4°C, the solution was diluted 1/5 in PBS and then dialyzed three times against 100 volumes of PBS at 4°C. The biotinylated sCD4 was compared with unlabelled sCD4 for the ability to bind to recombinant gpl20 or to HIV-infected cells and to induce gp4l exposure; the two forms of sCD4 were indistinguishable. (ii) Formation of sCD4-MAb complexes. Before being added to the HIV-1-infected cells, 25 ,ul of sCD4 at 88 nM was mixed with 25 p.1 of MAb or Fab in a 20- or 100-fold molar excess, respectively. The sCD4-MAb or sCD4-Fab complex was allowed to form for 30 min at roomn temperature before the addition of 50 p.1 of infected cells at 2 x 107 per ml and warming to 37°C. (iii) Infected cells. Cells were infected 10 days prior to use with either HIV-1 IIIB or HIV-1 RF at a multiplicity of infection of approximately 0.1. RESULTS Inhibition by CD4 MAbs of HIV infection and syncytium formation. We tested whether preincubation with the CD4 MAbs 6H10 and 5A8 and 5A8 Fab inhibited the infection of C8166 CD4+ T cells by cell-free HIV-1 RF by measuring p24 production after 3 days of culture and syncytium formation on day 7 (Table 1). Judged by both criteria, 6H10 and 5A8 inhibited RF infection at virus input doses of 12.5 and 125 TCID50. The 5A8 Fab fragment also inhibited p24 production, albeit at much higher concentrations than the complete 5A8 antibody, but was ineffective at blocking syncytium formation. The D3 and D4 MAbs OKT4 and L120 were only weakly active at blocking RF infection compared with 6H10 and 5A8. In general, higher MAb concentrations were required at higher virus doses (but see Fig. 1 below), and no inhibition was observed at any MAb concentration that could be tested when the RF input dose was 1,250 TCID50 or greater.
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TABLE 1. Inhibition by CD4 MAbs of HIV infection and syncytium formationa Inhibition of syncytium MAb
6H10 5A8 5A8 Fab OKT4 L120
formation with RF at: 12.5 TCID50 125 TCID50
10-20 1-10 >1,200 500 80-170
60-300 3-20 >1,200 >500 > 170
Inhibition of p24 production
with RF at: 125 TCID50
12.5 TCID50
1-1 1-3 9-18 ND 40-> 170
1-10 1-20 290-580 ND 40->170
a Data shown are ranges of the median inhibitory MAb and 5A8 Fab concentrations (nanomolar) in each assay from four independent syncytium assays and two independent p24 experiments. ND, not done.
When 6H10 and 5A8 were added to C8166 cells 2 h after virus, higher (fivefold or more) MAb concentrations were required for inhibition of infection compared with those which were effective when the MAbs were incubated with the cells before virus addition (data not shown). Thus the MAbs both block the initial infection by cell-free virus and antagonize later progeny virus infection or cell-to-cell spread of HIV in the infected cultures. The relative importance of inhibition of virion versus cell-to-cell spread could not be exactly determined. We found that infectivity assays with cell-free virus (Table 1) were unsuitable for quantitative analysis of the action of CD4 MAbs because the highest MAb concentrations gave no detectable inhibition at viral doses of 1,250 TCID50 or higher. We therefore used a short-term syncytium inhibition assay measuring fusion between RF-infected H9 cells and uninfected C8166 CD4+ cells to assess whether MAbs 5A8 and 6H10 were active by a competitive mechanism or otherwise (Fig. 1). In principle, a MAb such as 6H10 that blocks the virus binding site on CD4 Dl (4) should be
competitive in its inhibitory action with respect to virus concentration, whereas MAb 5A8 mapping in D2 (4) should be noncompetitive. When the input concentration of RFinfected cells (i.e., the concentration of env glycoproteins) was varied at a fixed CD4+ target cell concentration, the inhibitory concentration of MAb 6H10 showed a clear de-
pendence on env protein input (Fig. 1). This is consistent with reversible, competitive inhibition. In contrast, the inhibitory concentration of MAb 5A8 was essentially independent of env input over the range tested. This suggests that 5A8 inhibits syncytium formation by a noncompetitive mechanism, provided that sufficient 5A8 is present to saturate cell surface CD4. Effect of CD4 MAbs on gpl2O-sCD4 binding. To assess whether the CD4 MAbs affected gpl20 binding to CD4, we initially used an ELISA format. None among MAbs 5A8, OKT4, and L120 affected the binding of sCD4 to gpl20 solubilized from RF with NP-40 (Fig. 2a and b). In contrast, MAb 6H10 potently inhibited the sCD4-gpl2O interaction, which is consistent with the known overlap of its epitope with the gpl20 binding site on the CDR-2 loop of CD4 Dl (4). It therefore seemed unlikely that the inhibitory action of 5A8 on HIV-1 infection could be attributed to any quantitative effect on gpl2O-CD4 binding, provided that results obtained with soluble gpl20 could be extrapolated to situations with intact virions. Effect of CD4 MAbs on sCD4 binding to RF virions and on gp120 dissociation. Taken together, the above results suggest that MAb 5A8 inhibits postbinding events necessary for HIV-1 fusion with CD4+ cells. To test this in model systems, we first investigated the effects of 5A8 on sCD4 binding to RF virions and its induction of gpl20 dissociation by using gel filtration and ELISA methods. We found that ternary complexes of gpl2O-sCD4-MAb could form on the virion surface with saturating concentrations of MAbs 5A8 and OKT4 but not with the Dl MAb 6H10 (Fig. 3). Data similar
50 40
90%) to the cell surface (Fig. 8A). However, some MAb F91-55 bound to the cell surface, suggesting that ternary complexes, i.e., MAb F91-55-sCD4gpl20, can form. Of the other MAbs tested, both 5A8 and its Fab fragment inhibited sCD4 binding to the cells by about 50%, although ternary complexes could be clearly demonstrated. Explanations may be that this MAb reduces the affinity of sCD4 for gpl20 on the surface of infected cells, thus increasing dissociation, or prevents multiple sCD4 molecules from binding to a gpl20 oligomer (see also Fig. 6). The extent of this inhibition is dependent on MAb concentration, since at higher sCD4/MAb ratios little or no inhibition of sCD4 binding to the cells was observed, although inhibition of gp4l exposure still occurred (data not shown). This was not the case for MAb F91-55, which substantially reduced sCD4 binding to the cells at all concentrations that inhibited gp4l exposure. Neither OKT4 nor L120 inhibited sCD4 binding; indeed, L120 enhanced sCD4 binding to the cells by about 20%. 5A8, OKT4, and L120 all formed ternary complexes with sCD4 and gpl20 as demonstrated by staining the cell surface with anti-mouse phycoerthrin (Fig. 8A). We were unable to detect binding of 5A8 Fab fragment to cell-bound sCD4; since this Fab fragment was inhibitory for the indirectly detected sCD4-induced conformational changes, we assume that a ternary complex is formed between sCD4, gpl20, and 5A8 Fab but that our detection antibody was unable to recognize the Fab portion of 5A8. sCD4-induced gp4l exposure, as detected by the anti-gp4l MAbs 50-69 and 98-6, was substantially reduced by MAbs 6H10, F91-55, and 5A8 and by the 5A8 Fab fragment (Fig. 8B). OKT4 had no effect on gp4l exposure, whereas L120 had a small but reproducible inhibitory effect consistent with its weak inhibition of gpl20 shedding from virions (Fig. Sb). Thus 6H10 clearly inhibits gp4l exposure by competitive inhibition of sCD4 binding to gpl20, whereas 5A8 and its Fab fragment do so by another mechanism. MAb F91-55 appears to block gp4l exposure by a combination of competition and another mechanism, whereas OKT4 and L120 have little or
J. VIROL.
no effect. Results very similar to those described above were obtained for the action of these MAbs on sCD4-induced gp4l exposure on RF-infected cells (data not shown), demonstrating that the effect is not restricted to one viral genotype. For technical reasons relating to the murine nature of MAb 5A8, we have not yet been able to test its effect on sCD4-induced alteration of V3 loop epitopes. DISCUSSION The data described here indicate that the CD4 D2 MAb 5A8 and the CD4 Dl MAb 6H10 block HIV infection of CD4+ cells by distinct mechanisms. 6H10, like MAbs such as Leu3A, OKT4A, and Q4120, which bind to overlapping or contiguous Dl epitopes (4, 17, 37), is presumed to act by competitively inhibiting virus-cell attachment. In the absence of a binding assay of sufficient sensitivity to detect virus binding to cells at virus input concentrations appropriate for infectivity assays, we cannot demonstrate directly that 6H10 acts by blocking HIV binding to cell surface CD4 and that 5A8 does not. But data from our model systems suggest that 5A8 is very unlikely to act as a simple inhibitor of virus binding. Thus 5A8 did not inhibit soluble gpl20 binding to CD4+ cells (4) or sCD4 binding to soluble gpl20 (Fig. 2) and had only a small effect on sCD4 binding to virions and to HIV-1-infected cells (Fig. 5, 6, and 8). Ternary complexes of 5A8-sCD4-gpl20 could be detected on virions and HIV-1-infected cells (Fig. 3 and 8A). Finally, inhibition of env-mediated syncytium formation by 5A8 was not competitive with regard to env input (Fig. 1). Each of these observations contrasts with the data for 6H10. We believe, therefore, that virions bind to cell surface CD4 in the presence of 5A8 but that their fusion with the cell membrane is blocked. An analogous mechanism can be presumed to account for 5A8's inhibition of syncytium formation; when 5A8 was added to CD4-expressing cells prebound to cells expressing gpl20-gp41 for 1 h at 4°C (at which temperature fusion does not occur), both cell-cell fusion, monitored by fluorescent dye redistribution, and syncytium formation were inhibited when the cells were subsequently warmed to 37°C (lOa). Thus, 5A8 is affecting a stage of the fusion reaction occurring after the initial gpl20-CD4 interaction. The route between virus binding and pH-independent fusion is poorly understood for HIV, but it has been suggested that binding of HIV to CD4 directly activates the fusogenic potential of the virus by the induction of conformational changes in its envelope, and perhaps in CD4 as well (1, 2, 17, 21, 35, 38). We propose that 5A8 blocks these changes. The evidence for this is derived from model systems with sCD4, and extrapolations to the cellular system must be made with caution. Nonetheless, we observe that 5A8 blocks sCD4-induced dissociation of gpl20 from RF and IIIB virions and that it prevents exposure of cryptic epitopes on gp4l. 5A8 does not, however, inhibit sCD4-induced increase in cleavage of the gpl20 V3 region by the exogenous serine-proteinase thrombin. There may be a sequence of conformational changes in gpl20/gp4l after CD4 binding, only some of which are inhibited by 5A8. The blockage of conformational changes by 5A8 and its monovalent Fab fragment is paradoxical, since Dl alone is sufficient to induce gpl20 shedding (15, 36) and the 5A8 epitope is within D2 (4). One possibility is that the mere presence of 5A8 or 5A8 Fab on D2 hinders a conformational change induced by Dl or within Dl that is necessary for shedding. Another is that the D2 MAb interferes sterically with the function of regions of Dl distinct from the primary
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Mean fluorescence intensity FIG. 8. sCD4 binding to HIV-1-infected cells and gp4l exposure. H9 cells infected with HIV-1 (IIIB) were treated with biotinylated sCD4 biotinylated sCD4 complexed with the CD4 MAbs listed in the legend to Fig. 7 for 2 h at 37°C. After being washed, the cells were stained with streptavidin-phycoerythrin to measure bound sCD4 (O) or anti-mouse IgG phycoerythrin to measure MAb complexed with sCD4 (U) (a) and anti-gp4l MAbs 50-69 (0) and 98-6 (5), which recognize residues 579 to 613 and 644 to 663, respectively (b) (43). Staining of the infected cells in the absence of sCD4 is represented by a dotted line for 50-69 and a dashed line for 98-6. The stained cells were fixed in 1% formaldehyde and then analyzed on a Becton Dickinson FACScan. The results are expressed as the mean fluorescence intensity, where the background mean fluorescence (second-layer antibody only) has been subtracted from the test mean fluorescence. or
virus binding site (for example, the CDR3 region) that might be involved in postbinding events. The binding site for 5A8 on D2 is on the same face of CD4 as the CDR3 region on Dl (4), which has been proposed to be involved in gp120 binding (24). Although it is improbable, in our view, that CDR3 contributes significantly to the gpl20-CD4 binding energy, this region has been suggested to function during fusion (5, 41) and this function might be antagonized by 5A8. The binding of MAb L71 to CDR3 (41) is not, however, blocked by 5A8 (38a). 5A8 does reduce the binding of sCD4 to virions and
HIV-1-infected cells (Fig. 6 and 8) but not to soluble gp120 (Fig. 2). This implies that the effect is restricted to oligomeric gpl20 and is not directly on the gpl20-combining site. A possible explanation is that the bulkiness of 5A8 prevents binding of multiple sCD4 molecules onto a single glycoprotein oligomer, which is consistent with the observation of a twofold reduction in the extent of sCD4 binding to oligomeric gpl20 in the presence of 5A8 (Fig. 8A). We have reported that multiple occupancy of an oligomer is necessary for sCD4-induced gp120 shedding (34); such an indirect effect of 5A8 on sCD4 binding could therefore account for its
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blockade of shedding. By inference, 5A8 could also prevent clustering of sufficient CD4 molecules around a virion to form a fusion complex (23). Another, related, possibility is that 5A8 might hinder a conformational change that increases the affinity of env-glycoprotein oligomers for sCD4. Such conformational changes have been suggested to account for the temperature-dependent increase in the sCD4virion interaction and to be coupled to the induction of gpl20 dissociation (32), although it is uncertain whether temperature influences kinetic or thermodynamic parameters of sCD4 binding in this system (12, 19). Cross-linking of sCD4 by 5A8 may contribute to the reduction in sCD4 binding to virions (Fig. 6), since monovalent 5A8 Fab had a much reduced effect on sCD4 binding compared with 5A8 while the D4 MAb L120 also had a weak inhibitory effect in some experiments, although OKT4 did not. The interactions are clearly complex, however, since both 5A8 and the Fab fragment reduced sCD4 binding to infected cells, whereas L120 increased binding (Fig. 8A). Bivalency might be expected to increase the functional affinity by an avidity effect, as for the higher affinity of the interaction between virions and bivalent CD4-Ig chimeras (33). But a 5A8-sCD4 complex is unlikely to have the same gp120 combining site orientation as on CD4-Ig, so the analogy may not be exact. Cross-linking might contribute to the inhibitory action of 5A8 on gpl20 shedding and gp4l although it cannot be the only operative mechanism, as 5A8 Fab also blocked this. It is notable, however, that 5A8 Fab inhibits infectivity by cell-free HIV-1 although it does not inhibit env-induced syncytium formation (Table 1) (4). The latter requires a much larger number of gpl20CD4 interactions than are needed for virus-cell fusion (11) and is also relatively resistant to inhibition by sCD4 (9, 12). Thus the inhibitory effect on HIV-mediated syncytium formation, which 5A8 has and its Fab lacks (Table 1) (4), could be dependent on a combination of cross-linking of CD4 molecules and steric interference by the Fc portion of the antibody. We do not know how important the particular conformational changes we infer from model systems are for HIV infectivity and syncytium formation. Shedding of gp120, which is blocked by 5A8, may not be necessary for fusion and might indeed abort the fusion reaction prematurely (1, 35). Nor do we know how exposure of previously occult gp4l epitopes influences fusion. However, we suggest that these conformational changes at least partially mimic those happening in the real fusion reaction and that their antagonism by 5A8 accounts for its inhibition of HIV-1 infection. The D3 MAb, Q425 (17), also blocks HIV infectivity at a stage after binding. Its action appears in preliminary experiments to be similar to that of 5A8 but not identical (38a). Furthermore, Q425 and its Fab fragment block sCD4 enhancement of HIV-2 fusion with CD4 cells (8), whereas 5A8 does not (6a). Together, 5A8, Q425, and related MAbs might allow us to dissect further the stages of the HIV-cell fusion reaction.
exposure,
ACKNOWLEDGMENTS
We are grateful to Robin Weiss and David Ho for provision of facilities and for advice. We thank Kathleen Kimball and Renee Shapiro for preparing MAbs and Fab fragments and Dian Olson and Wendy Chen for help in preparing the figures. We are grateful to Susan Zolla-Pazner for MAbs 50-69 and 98-6 and to Franco Celada for MAb F91-55. We also appreciate Paul Clapham's and Thomas Schulz's critical comments on the manuscript and permission from Mitko Dimitrov to quote unpublished results.
This work was funded by the AIDS Directed Programme of the UK Medical Research Council; the Cancer Research Campaign; The Aaron Diamond Foundation; and Biogen, Inc. P.J.K. is a beneficiary of a scholarship from the Medical Faculty, University of Lund, Lund, Sweden. REFERENCES 1. Allan, J. S. 1991. Receptor-mediated activation of immunodeficiency viruses in viral fusion. Science 252:1322. 2. Allan, J. S., J. Strauss, and D. W. Buck. 1990. Enhancement of SIV infection with soluble receptor molecules. Science 247: 1084-1088. 3. Ashorn, P. A., E. A. Berger, and B. Moss. 1990. Human immunodeficiency virus envelope glycoprotein/CD4-mediated fusion of nonprimate cells with human cells. J. Virol. 64:21492156. 4. Burkly, L. C., D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm. Inhibition of HIV entry by a CD4 domain 2 specific monoclonal antibody: evi-
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Antibody raised against soluble CD4-rgpl2O complex recognizes the CD4 moiety and blocks membrane fusion without inhibiting CD4-gpl20 binding. J. Exp. Med. 172:1143-1154. 6a.Clapham, P. R. Personal communication. 7. Clapham, P. R., D. Blanc, and R. A. Weiss. 1991. Specific cell surface requirements for infection of CD4-positive cells by human immunodeficiency virus type 1, type 2 and simian immunodeficiency virus. Virology 181:703-715. 8. Clapham, P. R., A. McKnight, and R. A. Weiss. 1992. Human immunodeficiency virus type 2 infection and fusion of CD4negative human cell lines: induction and enhancement by soluble CD4. J. Virol. 66:3531-3537. 9. Clapham, P. R., J. N. Weber, D. Whitby, K. McIntosh, A. G. Dalgleish, P. J. Maddon, K. C. Deen, R. W. Sweet, and R. A.
Weiss. 1989. Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells. Nature (London) 337:368-370. 10. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4)
antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312:763-766. 10a.Dimitrov. D. S. Personal communication. 11. Dimitrov, D. S., H. Golding, and R. Blumenthal. 1991. Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion
monitored by a new assay based on redistribution of fluorescent dyes. AIDS Res. Hum. Retroviruses 7:799-805. 12. Dimitrov, D. S., K. Hillman, J. Manischewitz, R. Blumenthal,
and H. Golding. 1992. Kinetics of soluble CD4 binding to cells expressing human immunodeficiency virus type 1 envelope
glycoprotein. J. Virol. 66:132-138. 13. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch,
R. T. Schooley, and R. A. Flavell. 1988. HIV infection is blocked in vitro by recombinant soluble CD4. Nature (London) 331: 7678. 14. Grewe, C., A. Beck, and H. R. Gelderblom. 1990. HIV: early virus-cell interactions. J. Acquired Immune Defic. Syndr. 3:965-974. 15. Hart, T. K., R. Kirsh, H. Ellens, R. W. Sweet, D. M. Lambert, S. R. Pettaway, J. Leary, and P. Bugelski. 1991. CD4-HIV-1 interactions: binding of soluble CD4 (sT4) to HIV-1 and HIV-1 infected cells induces shedding of envelope gpl20. Proc. Natl. Acad. Sci. USA 88:2189-2193. 16. Hasunuma, T., H. Tsubota, M. Watanabe, Z. W. Chen, C. I. Lord, L. C. Burkly, J. F. Daley, and N. L. Letvin. 1992. Regions
of the CD4 molecule not involved in virus binding or syncytia formation are required for HIV-1 infection of lymphocytes. J.
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Immunol. 148:1841-1846. 17. Healey, D., L. Dianda, J. P. Moore, J. S. McDougal, M. J. Moore, P. Estess, D. Buck, P. D. Kwong, P. C. L. Beverley, and Q. J. Sattentau. 1990. Novel anti-CD4 monoclonal antibodies separate HIV infection and fusion of CD4+ cells from virus binding. J. Exp. Med. 172:1233-1242. 18. Kirsh, R., T. K. Hart, H. Ellens, J. Miller, S. R. Petteway, Jr., D. M. Lambert, J. Leary, and P. J. Bugelskd. 1990. Morphometric analysis of recombinant soluble CD4 mediated release of the envelope glycoprotein gpl20 from HIV-1. AIDS Res. Hum. Retroviruses 6:1209-1212. 19. Klasse, P. J., and J. P. Moore. 1992. Kinetics of the HIV-CD4 interaction and virus-cell fusion. AIDS 6:325-327. 20. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.-C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312:767-768. 21. Kowalski, M. L., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:13511355. 22. Lasky, L. A., G. M. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. W. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodeficiency virus type 1 gpl20 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. 23. Layne, S. P., M. J. Merges, M. Dembo, J. L. Spouge, and P. L. Nara. 1990. HIV requires multiple gpl20 molecules for CD4mediated infection. Nature (London) 346:277-279. 24. Lifson, J. D., D. M. Rausch, V. S. Kalyanaraman, K. M. Hwang, and L. E. Eiden. 1991. Synthetic peptides allow discrimination of structural features of CD4(81-92) important for HIV-1 infection versus HIV-1 induced syncytium formation. AIDS Res. Hum. Retroviruses 7:521-528. 25. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and brain. Cell 47:333-348. 26. McClure, M. O., M. Marsh, and R. A. Weiss. 1988. Human immunodeficiency virus infection of CD4 bearing cells occurs by a pH independent mechanism. EMBO J. 7:513-518. 27. McDougal, J. S., M. S. Kennedy, J. M. Sligh, S. P. Cort, A. Mawle, and J. K. A. Nicholson. 1986. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. Science 231:382-385. 28. McDougal, J. S., J. K. A. Nicholson, G. D. Cross, M. S. Kennedy, and A. C. Mawle. 1986. Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformational dependence, epitope mapping, antibody inhibition and potential for idiotypic mimicry. J. Immunol. 137:29372944. 29. McKeating, J. A., A. McKnight, and J. P. Moore. 1991. Differential loss of envelope glycoprotein gpl20 from virions of human immunodeficiency virus type 1 isolates: effect on infectivity and neutralization. J. Virol. 65:852-860. 30. Moore, J. P. 1990. Simple methods for monitoring HIV-1 and
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HIV-2 gp2O binding to sCD4 by ELISA: HIV-2 has a 25-fold lower affinity than HIV-1 for sCD4. AIDS 4:297-305. 30a.Moore, J. P. Unpublished data. 31. Moore, J. P., B. A. Jameson, R. A. Weiss, and Q. J. Sattentau. The HIV-cell fusion reaction. In J. Bentz (ed.), Viral fusion mechanisms, in press. CRC Press, Inc., Boca Raton, Fla. 32. Moore, J. P., and P. J. Klasse. 1992. Thermodynamic and kinetic analysis of sCD4 binding to HIV-1 virions and of gpl20 dissociation. AIDS Res. Hum. Retroviruses 8:443-450. 33. Moore, J. P., J. A. McKeating, Y. Huang, A. Ashkenazi, and D. D. Ho. 1992. Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gpl20 retention from sCD4-sensitive isolates. J. Virol. 66:235-243. 34. Moore, J. P., J. A. McKeating, W. A. Norton, and Q. J. Sattentau. 1991. Direct measurement of soluble CD4 binding to human immunodeficiency virus type 1 virions: gpl20 dissociation and its implications for virus-cell binding and fusion reactions and their neutralization by soluble CD4. J. Virol. 65:11331140. 35. Moore, J. P., J. A. McKeating, R. A. Weiss, P. R. Clapham, and Q. J. Sattentau. 1991. Receptor-mediated activation of immunodeficiency viruses in viral fusion. Science 252:1322-1323. 36. Moore, J. P., J. A. McKeating, R. A. Weiss, and Q. J. Sattentau. 1990. Dissociation of gpl20 from HIV-1 virions induced by soluble CD4. Science 250:1139-1142. 37. Sattentau, Q. J., J. Arthos, K. Deen, N. Hanna, D. Healey, P. C. L. Beverley, R. Sweet, and A. Truneh. 1989. Structural analysis of the human immunodeficiency virus binding domain of CD4. J. Exp. Med. 170:1319-1334. 38. Sattentau, Q. J., and J. P. Moore. 1991. Conformational changes induced in the human immunodeficiency virus envelope glycoproteins by soluble CD4 binding. J. Exp. Med. 174:407-415. 38a.Sattentau, Q. J., et al. Unpublished data. 39. Stein, B., S. Gowda, J. Lifson, R. Penhallow, K. Bensch, and E. Engelman. 1987. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell 49:659-668. 40. Thali, M., U. Olshevsky, C. Furman, D. Gabuzda, J. Li, and J. Sodroski. 1991. Effects of changes in gpl2O-CD4 binding affinity on human immunodeficiency virus type 1 envelope glycoprotein function and soluble CD4 sensitivity. J. Virol. 65:5007-5012. 41. Truneh, A., D. Buck, D. R. Cassatt, R. Juszczak, S. Kassis, S. E. Ryu, D. Healey, R. Sweet, and Q. Sattentau. 1991. A region in domain 1 of CD4 distinct from the primary gpl20 binding site is involved in HIV infection and virus mediated fusion. J. Biol. Chem. 266:5942-5948. 42. Watanabe, M., Z. W. Chen, H. Tsubota, C. I. Lord, C. G. Levine, and N. L. Letvin. 1991. Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication. Proc. Natl. Acad. Sci. USA 88:4616-4620. 43. Xu, J.-Y., M. K. Gorny, T. Palker, S. Karwowska, and S. Zolla-Pazner. 1991. Epitope mapping of two immunodominant domains of gp4l, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies. J. Virol. 65:4832-4838.
Vol. 66, No. 8
0022-538X192/084784-10$02.00/0 Copyright © 1992, American Society for Microbiology
A Monoclonal Antibody to CD4 Domain 2 Blocks Soluble CD4-Induced Conformational Changes in the Envelope Glycoproteins of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-1 Infection of CD4+ Cells JOHN P. MOORE,l,2* QUENTIN J. SATTENTAU,3 P. J. KLASSE,2 AND LINDA C. BURKLY4
Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom2; The Aaron Diamond AIDS Research Center, 455 First Avenue, New York, New York 100161*; Centre d'Immunologie de Marseille-Luminy, 13288 Marseille Cede-x 9, France3; and Biogen Inc., Cambridge, Massachusetts 021424 Received 19 March 1992/Accepted 6 May 1992
The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp4l and dissociation of gpl20 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gpl20/gp41 and/or CD4 that are necessary for virus-cell fusion.
pH-independent fusion of human immunodeficiency virus
proteinase (38); exposure of gp4l epitopes cryptic prior to sCD4 binding (38); and complete dissociation of gpl20 from gp4l (15, 18, 34, 36). Whether any or all of these changes are relevant to the virus-cell fusion process has been a matter for debate (1, 31, 35). In this study, we have tested the effect of anti-CD4 monoclonal antibodies (MAbs) on the conformational changes outlined above. One of these MAbs, 5A8, which binds to D2, potently inhibits HIV-1 infection without blocking gpl20 binding to CD4+ cells (4). Here we report evidence that 5A8 blocks sCD4-induced conformational changes in the HIV envelope glycoproteins. We suggest that 5A8, and other MAbs of this type (6, 16, 17), might block HIV infection by inhibiting similar reactions necessary for viruscell fusion. Our observations might also be relevant to the mechanism of action of protective polyclonal antibodies to simian CD4 raised in rhesus monkeys immunized with human sCD4 (42).
type 1 (HIV-1) with the plasma membrane of CD4+ cells is initiated by the binding of virions to domain 1 (D1) of CD4
(10, 20, 22, 26-28, 39; for a review, see reference 31). The mechanism of the fusion reaction is not known in any detail for HIV-1, or indeed for any retrovirus, but we and others have suggested that the binding of the HIV-1 surface (SU) glycoprotein gpl20 to CD4 induces conformational changes in the envelope glycoproteins and in CD4 (1, 2, 6, 17, 21, 35, 38). These molecular rearrangements are presumed to be necessary to establish an interaction of the fusion domain at the amino terminus of the transmembrane (TM) glycoprotein gp4l with the target cell membrane. Accessory cell surface factors seem likely to be involved at some stage of the fusion process, but their nature and role are at present obscure (3, 7, 25). Binding of HIV-1 virions to CD4+ cells can be detected biochemically (27, 28) and by electron microscopy (14), and fusing virions can be observed by the latter technique (14). HIV-1 cell fusion has also been monitored by a dye redistribution assay (11). However, there are at present no methods available to study conformational changes occurring during the interaction of virions with cell surface CD4. To gain some insights into the fusion reaction, we have therefore studied the interaction of soluble CD4 (sCD4) with HIV-1 virions and the surface of HIV-1-infected cells. With these model systems, we can obtain evidence that suggests the occurrence of conformational changes. This evidence includes temperature-dependent increases in the binding of sCD4 to virion-bound gpl20, which is consistent with a complex binding reaction (12, 32, 34, 40); sCD4-induced increases in the binding of antibodies to the V3 region of gp120 and its cleavage by an exogenous *
MATERIALS AND METHODS Antibodies and sCD4. MAbs 6H10 (D1), 5A8 (D2), F91-55 (D2), OKT4 (D3), and L120 (D4), mapping to the CD4 domains listed in parentheses, have all been described elsewhere (4, 6, 17). MAb 5A8 recognizes an epitope in CD4 D2 with the critical residues being between 121 and 134, as defined by chimeras of mouse and human CD4 and CD4 substitution mutants (4). The human anti-gp4l MAbs 50-69 and 98-6 were kind gifts from S. Zolla-Pazner. MAb 50-69 recognizes a peptide with residues 579 to 613, provided that Cys-598 and -604 are disulfide linked (43). MAb 98-6 competes for binding to gp4l with a MAb that recognizes a peptide with the sequence 644 to 663 (43). All MAbs, and Fab fragments of 5A8 made by conventional techniques (4), were used as purified immunoglobulins. Streptavidin-phyco-
Corresponding author. 4784
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erythrin and anti-mouse phycoerythrin were purchased from Immunotech S.A. (Marseille, France). sCD4 produced as previously described (13) was from Biogen Inc. (Cambridge, Mass.). V'iruses. Virus-containing supernatants of the cell cultureadapted HIV-1 isolates HTLV-III RF (RF) and HTLV-IIIB (IIIB) were obtained by cocultivation of infected and uninfected H9 cells as previously described (29). Virus titers for the stocks used in these experiments were typically 106 to 107 50% tissue culture infective doses (TCID50) per ml. Aliquots were stored under liquid nitrogen and each was used once only. Infectivity assays. The ability of CD4 MAbs to inhibit infection of C8166 cells by RF was assessed by incubating the MAbs (in triplicate) at a range of concentrations (1 to 500 nM, or 9 to 1,200 nM for 5A8 Fab) with 2 x 104 cells in 100 ,ul of RPMI 1640 medium-10% fetal calf serum for 20 min at room temperature. RF (12.5 to 125,000 TCID50 per 50 ,ul) was then added and the plates were incubated at 37°C in the presence of 5% CO2. After 3 days, aliquots (50 ,ul) of supernatants were collected, inactivated with 1% Empigen detergent, and analyzed for p24 content (36). After 7 days, syncytia were scored by light microscopy. The median MAb concentrations giving complete inhibition of syncytium formation or 99% inhibition) were recorded. To distinguish between inhibition of the initial infection by cell-free virus in the inoculum and inhibition of subsequent viral spread by cell-to-cell fusion or infection by progeny virus, the above assay was compared with one in which the cells were first incubated with the virus for 2 h at 37°C and then the MAbs were added. A weaker effect of the antibodies in the latter than in the former assay would be evidence of inhibition of initial infection. Syncytium inhibition assay. H9 cells were infected with RF at a multiplicity of infection of approximately 0.2. After 3 to 4 days of culture, they were washed once in RPMI 1640 medium, resuspended at 2 x 105 cells per ml, and diluted serially in eight twofold steps. 5A8 or 6H10 was diluted in twofold steps and incubated at final concentrations from 67 nM with 4 x 105 C8166 cells per ml in 96-well culture plates (100 p.1 per well) for 20 min at room temperature. A range of dilutions of RF-infected H9 cells (50 ,u1 per well) was then added, and the cocultures were incubated for 24 h at 37°C. The wells were examined by light microscopy for the presence of syncytia. gpl2O-sCD4 enzyme-linked immunosorbent assay (ELISA). RF virus in RPMI 1640 medium containing 10% fetal calf serum was disrupted with 1% Nonidet P-40 (NP-40) (RPMINP-40) to provide a source of soluble gpl20. This was incubated at 50 pM gpl20 per sample with preformed MAbsCD4 complexes in a total volume of 100 p.1 for 1 h at 37°C. The mixtures were supplemented with 2% nonfat milk powder (Marvel; Cadbury Ltd.) and gpl2O-sCD4 complexes (+ bound MAb) were then captured onto a solid phase by adsorbed antibody D7324 to the carboxy terminus of gpl20
(30). Bound sCD4 was detected with a cocktail of MAbs 5A8, L120, and OKT4, each at a saturating concentration of 0.3 ,ug/ml, followed by alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulin G (IgG) (RaMIg-AP). Datum points are the means of triplicate determinations, with standard deviations typically within 10% of the means if not shown. sCD4 binding to virions and gpl20 dissociation. sCD4 binding to intact virions was assessed by a combination of
4785
gel filtration and ELISA (34, 36). Briefly, virions incubated with sCD4 and/or MAbs were separated from unbound sCD4 and MAbs by gel filtration on Sephacryl S-1000 and then disrupted with 1% NP-40. gpl20 was captured onto a solid phase by antibody D7324. Bound gpl20 was then detected with a pool of HIV+ human sera and alkaline phosphataseconjugated goat anti-human Ig. Alternatively, sCD4-gpl2O complexes were detected with an appropriate pool of antiCD4 MAbs and RaMIg-AP. In some experiments, virionbound MAb was detected with RaMIg-AP after capture of gpl20 onto the solid phase as described above. Note that in this assay, signals can be obtained only if a ternary complex of MAb-sCD4-gpl2O forms on the virion prior to gel filtration. Proteolytic cleavage of the gp120 V3 loop. Thrombin cleavage of gpl20 into 70- and 50-kDa fragments was measured by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) as described previously (38). Exposure of gp4l epitopes. sCD4-induced epitope modulation was measured by a previously described method (38), with the following modifications. (i) Biotinylation of sCD4. In order to detect sCD4 precomplexed with CD4 MAb and bound to the surface of infected cells, sCD4 was biotinylated by the following procedure. Twenty microliters of a solution of sCD4 (130 p.M) in Ca2+and Mg2+-free phosphate-buffered saline (PBS), was diluted sixfold in 100 mM sodium hydrogen carbonate buffer at pH 8.5 and rapidly mixed with 80 p.g of NHS-LC-biotin (Pierce, Rockford, Ill.) per ml. After 24 h of incubation at 4°C, the solution was diluted 1/5 in PBS and then dialyzed three times against 100 volumes of PBS at 4°C. The biotinylated sCD4 was compared with unlabelled sCD4 for the ability to bind to recombinant gpl20 or to HIV-infected cells and to induce gp4l exposure; the two forms of sCD4 were indistinguishable. (ii) Formation of sCD4-MAb complexes. Before being added to the HIV-1-infected cells, 25 ,ul of sCD4 at 88 nM was mixed with 25 p.1 of MAb or Fab in a 20- or 100-fold molar excess, respectively. The sCD4-MAb or sCD4-Fab complex was allowed to form for 30 min at roomn temperature before the addition of 50 p.1 of infected cells at 2 x 107 per ml and warming to 37°C. (iii) Infected cells. Cells were infected 10 days prior to use with either HIV-1 IIIB or HIV-1 RF at a multiplicity of infection of approximately 0.1. RESULTS Inhibition by CD4 MAbs of HIV infection and syncytium formation. We tested whether preincubation with the CD4 MAbs 6H10 and 5A8 and 5A8 Fab inhibited the infection of C8166 CD4+ T cells by cell-free HIV-1 RF by measuring p24 production after 3 days of culture and syncytium formation on day 7 (Table 1). Judged by both criteria, 6H10 and 5A8 inhibited RF infection at virus input doses of 12.5 and 125 TCID50. The 5A8 Fab fragment also inhibited p24 production, albeit at much higher concentrations than the complete 5A8 antibody, but was ineffective at blocking syncytium formation. The D3 and D4 MAbs OKT4 and L120 were only weakly active at blocking RF infection compared with 6H10 and 5A8. In general, higher MAb concentrations were required at higher virus doses (but see Fig. 1 below), and no inhibition was observed at any MAb concentration that could be tested when the RF input dose was 1,250 TCID50 or greater.
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TABLE 1. Inhibition by CD4 MAbs of HIV infection and syncytium formationa Inhibition of syncytium MAb
6H10 5A8 5A8 Fab OKT4 L120
formation with RF at: 12.5 TCID50 125 TCID50
10-20 1-10 >1,200 500 80-170
60-300 3-20 >1,200 >500 > 170
Inhibition of p24 production
with RF at: 125 TCID50
12.5 TCID50
1-1 1-3 9-18 ND 40-> 170
1-10 1-20 290-580 ND 40->170
a Data shown are ranges of the median inhibitory MAb and 5A8 Fab concentrations (nanomolar) in each assay from four independent syncytium assays and two independent p24 experiments. ND, not done.
When 6H10 and 5A8 were added to C8166 cells 2 h after virus, higher (fivefold or more) MAb concentrations were required for inhibition of infection compared with those which were effective when the MAbs were incubated with the cells before virus addition (data not shown). Thus the MAbs both block the initial infection by cell-free virus and antagonize later progeny virus infection or cell-to-cell spread of HIV in the infected cultures. The relative importance of inhibition of virion versus cell-to-cell spread could not be exactly determined. We found that infectivity assays with cell-free virus (Table 1) were unsuitable for quantitative analysis of the action of CD4 MAbs because the highest MAb concentrations gave no detectable inhibition at viral doses of 1,250 TCID50 or higher. We therefore used a short-term syncytium inhibition assay measuring fusion between RF-infected H9 cells and uninfected C8166 CD4+ cells to assess whether MAbs 5A8 and 6H10 were active by a competitive mechanism or otherwise (Fig. 1). In principle, a MAb such as 6H10 that blocks the virus binding site on CD4 Dl (4) should be
competitive in its inhibitory action with respect to virus concentration, whereas MAb 5A8 mapping in D2 (4) should be noncompetitive. When the input concentration of RFinfected cells (i.e., the concentration of env glycoproteins) was varied at a fixed CD4+ target cell concentration, the inhibitory concentration of MAb 6H10 showed a clear de-
pendence on env protein input (Fig. 1). This is consistent with reversible, competitive inhibition. In contrast, the inhibitory concentration of MAb 5A8 was essentially independent of env input over the range tested. This suggests that 5A8 inhibits syncytium formation by a noncompetitive mechanism, provided that sufficient 5A8 is present to saturate cell surface CD4. Effect of CD4 MAbs on gpl2O-sCD4 binding. To assess whether the CD4 MAbs affected gpl20 binding to CD4, we initially used an ELISA format. None among MAbs 5A8, OKT4, and L120 affected the binding of sCD4 to gpl20 solubilized from RF with NP-40 (Fig. 2a and b). In contrast, MAb 6H10 potently inhibited the sCD4-gpl2O interaction, which is consistent with the known overlap of its epitope with the gpl20 binding site on the CDR-2 loop of CD4 Dl (4). It therefore seemed unlikely that the inhibitory action of 5A8 on HIV-1 infection could be attributed to any quantitative effect on gpl2O-CD4 binding, provided that results obtained with soluble gpl20 could be extrapolated to situations with intact virions. Effect of CD4 MAbs on sCD4 binding to RF virions and on gp120 dissociation. Taken together, the above results suggest that MAb 5A8 inhibits postbinding events necessary for HIV-1 fusion with CD4+ cells. To test this in model systems, we first investigated the effects of 5A8 on sCD4 binding to RF virions and its induction of gpl20 dissociation by using gel filtration and ELISA methods. We found that ternary complexes of gpl2O-sCD4-MAb could form on the virion surface with saturating concentrations of MAbs 5A8 and OKT4 but not with the Dl MAb 6H10 (Fig. 3). Data similar
50 40
90%) to the cell surface (Fig. 8A). However, some MAb F91-55 bound to the cell surface, suggesting that ternary complexes, i.e., MAb F91-55-sCD4gpl20, can form. Of the other MAbs tested, both 5A8 and its Fab fragment inhibited sCD4 binding to the cells by about 50%, although ternary complexes could be clearly demonstrated. Explanations may be that this MAb reduces the affinity of sCD4 for gpl20 on the surface of infected cells, thus increasing dissociation, or prevents multiple sCD4 molecules from binding to a gpl20 oligomer (see also Fig. 6). The extent of this inhibition is dependent on MAb concentration, since at higher sCD4/MAb ratios little or no inhibition of sCD4 binding to the cells was observed, although inhibition of gp4l exposure still occurred (data not shown). This was not the case for MAb F91-55, which substantially reduced sCD4 binding to the cells at all concentrations that inhibited gp4l exposure. Neither OKT4 nor L120 inhibited sCD4 binding; indeed, L120 enhanced sCD4 binding to the cells by about 20%. 5A8, OKT4, and L120 all formed ternary complexes with sCD4 and gpl20 as demonstrated by staining the cell surface with anti-mouse phycoerthrin (Fig. 8A). We were unable to detect binding of 5A8 Fab fragment to cell-bound sCD4; since this Fab fragment was inhibitory for the indirectly detected sCD4-induced conformational changes, we assume that a ternary complex is formed between sCD4, gpl20, and 5A8 Fab but that our detection antibody was unable to recognize the Fab portion of 5A8. sCD4-induced gp4l exposure, as detected by the anti-gp4l MAbs 50-69 and 98-6, was substantially reduced by MAbs 6H10, F91-55, and 5A8 and by the 5A8 Fab fragment (Fig. 8B). OKT4 had no effect on gp4l exposure, whereas L120 had a small but reproducible inhibitory effect consistent with its weak inhibition of gpl20 shedding from virions (Fig. Sb). Thus 6H10 clearly inhibits gp4l exposure by competitive inhibition of sCD4 binding to gpl20, whereas 5A8 and its Fab fragment do so by another mechanism. MAb F91-55 appears to block gp4l exposure by a combination of competition and another mechanism, whereas OKT4 and L120 have little or
J. VIROL.
no effect. Results very similar to those described above were obtained for the action of these MAbs on sCD4-induced gp4l exposure on RF-infected cells (data not shown), demonstrating that the effect is not restricted to one viral genotype. For technical reasons relating to the murine nature of MAb 5A8, we have not yet been able to test its effect on sCD4-induced alteration of V3 loop epitopes. DISCUSSION The data described here indicate that the CD4 D2 MAb 5A8 and the CD4 Dl MAb 6H10 block HIV infection of CD4+ cells by distinct mechanisms. 6H10, like MAbs such as Leu3A, OKT4A, and Q4120, which bind to overlapping or contiguous Dl epitopes (4, 17, 37), is presumed to act by competitively inhibiting virus-cell attachment. In the absence of a binding assay of sufficient sensitivity to detect virus binding to cells at virus input concentrations appropriate for infectivity assays, we cannot demonstrate directly that 6H10 acts by blocking HIV binding to cell surface CD4 and that 5A8 does not. But data from our model systems suggest that 5A8 is very unlikely to act as a simple inhibitor of virus binding. Thus 5A8 did not inhibit soluble gpl20 binding to CD4+ cells (4) or sCD4 binding to soluble gpl20 (Fig. 2) and had only a small effect on sCD4 binding to virions and to HIV-1-infected cells (Fig. 5, 6, and 8). Ternary complexes of 5A8-sCD4-gpl20 could be detected on virions and HIV-1-infected cells (Fig. 3 and 8A). Finally, inhibition of env-mediated syncytium formation by 5A8 was not competitive with regard to env input (Fig. 1). Each of these observations contrasts with the data for 6H10. We believe, therefore, that virions bind to cell surface CD4 in the presence of 5A8 but that their fusion with the cell membrane is blocked. An analogous mechanism can be presumed to account for 5A8's inhibition of syncytium formation; when 5A8 was added to CD4-expressing cells prebound to cells expressing gpl20-gp41 for 1 h at 4°C (at which temperature fusion does not occur), both cell-cell fusion, monitored by fluorescent dye redistribution, and syncytium formation were inhibited when the cells were subsequently warmed to 37°C (lOa). Thus, 5A8 is affecting a stage of the fusion reaction occurring after the initial gpl20-CD4 interaction. The route between virus binding and pH-independent fusion is poorly understood for HIV, but it has been suggested that binding of HIV to CD4 directly activates the fusogenic potential of the virus by the induction of conformational changes in its envelope, and perhaps in CD4 as well (1, 2, 17, 21, 35, 38). We propose that 5A8 blocks these changes. The evidence for this is derived from model systems with sCD4, and extrapolations to the cellular system must be made with caution. Nonetheless, we observe that 5A8 blocks sCD4-induced dissociation of gpl20 from RF and IIIB virions and that it prevents exposure of cryptic epitopes on gp4l. 5A8 does not, however, inhibit sCD4-induced increase in cleavage of the gpl20 V3 region by the exogenous serine-proteinase thrombin. There may be a sequence of conformational changes in gpl20/gp4l after CD4 binding, only some of which are inhibited by 5A8. The blockage of conformational changes by 5A8 and its monovalent Fab fragment is paradoxical, since Dl alone is sufficient to induce gpl20 shedding (15, 36) and the 5A8 epitope is within D2 (4). One possibility is that the mere presence of 5A8 or 5A8 Fab on D2 hinders a conformational change induced by Dl or within Dl that is necessary for shedding. Another is that the D2 MAb interferes sterically with the function of regions of Dl distinct from the primary
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Mean fluorescence intensity FIG. 8. sCD4 binding to HIV-1-infected cells and gp4l exposure. H9 cells infected with HIV-1 (IIIB) were treated with biotinylated sCD4 biotinylated sCD4 complexed with the CD4 MAbs listed in the legend to Fig. 7 for 2 h at 37°C. After being washed, the cells were stained with streptavidin-phycoerythrin to measure bound sCD4 (O) or anti-mouse IgG phycoerythrin to measure MAb complexed with sCD4 (U) (a) and anti-gp4l MAbs 50-69 (0) and 98-6 (5), which recognize residues 579 to 613 and 644 to 663, respectively (b) (43). Staining of the infected cells in the absence of sCD4 is represented by a dotted line for 50-69 and a dashed line for 98-6. The stained cells were fixed in 1% formaldehyde and then analyzed on a Becton Dickinson FACScan. The results are expressed as the mean fluorescence intensity, where the background mean fluorescence (second-layer antibody only) has been subtracted from the test mean fluorescence. or
virus binding site (for example, the CDR3 region) that might be involved in postbinding events. The binding site for 5A8 on D2 is on the same face of CD4 as the CDR3 region on Dl (4), which has been proposed to be involved in gp120 binding (24). Although it is improbable, in our view, that CDR3 contributes significantly to the gpl20-CD4 binding energy, this region has been suggested to function during fusion (5, 41) and this function might be antagonized by 5A8. The binding of MAb L71 to CDR3 (41) is not, however, blocked by 5A8 (38a). 5A8 does reduce the binding of sCD4 to virions and
HIV-1-infected cells (Fig. 6 and 8) but not to soluble gp120 (Fig. 2). This implies that the effect is restricted to oligomeric gpl20 and is not directly on the gpl20-combining site. A possible explanation is that the bulkiness of 5A8 prevents binding of multiple sCD4 molecules onto a single glycoprotein oligomer, which is consistent with the observation of a twofold reduction in the extent of sCD4 binding to oligomeric gpl20 in the presence of 5A8 (Fig. 8A). We have reported that multiple occupancy of an oligomer is necessary for sCD4-induced gp120 shedding (34); such an indirect effect of 5A8 on sCD4 binding could therefore account for its
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MOORE ET AL.
blockade of shedding. By inference, 5A8 could also prevent clustering of sufficient CD4 molecules around a virion to form a fusion complex (23). Another, related, possibility is that 5A8 might hinder a conformational change that increases the affinity of env-glycoprotein oligomers for sCD4. Such conformational changes have been suggested to account for the temperature-dependent increase in the sCD4virion interaction and to be coupled to the induction of gpl20 dissociation (32), although it is uncertain whether temperature influences kinetic or thermodynamic parameters of sCD4 binding in this system (12, 19). Cross-linking of sCD4 by 5A8 may contribute to the reduction in sCD4 binding to virions (Fig. 6), since monovalent 5A8 Fab had a much reduced effect on sCD4 binding compared with 5A8 while the D4 MAb L120 also had a weak inhibitory effect in some experiments, although OKT4 did not. The interactions are clearly complex, however, since both 5A8 and the Fab fragment reduced sCD4 binding to infected cells, whereas L120 increased binding (Fig. 8A). Bivalency might be expected to increase the functional affinity by an avidity effect, as for the higher affinity of the interaction between virions and bivalent CD4-Ig chimeras (33). But a 5A8-sCD4 complex is unlikely to have the same gp120 combining site orientation as on CD4-Ig, so the analogy may not be exact. Cross-linking might contribute to the inhibitory action of 5A8 on gpl20 shedding and gp4l although it cannot be the only operative mechanism, as 5A8 Fab also blocked this. It is notable, however, that 5A8 Fab inhibits infectivity by cell-free HIV-1 although it does not inhibit env-induced syncytium formation (Table 1) (4). The latter requires a much larger number of gpl20CD4 interactions than are needed for virus-cell fusion (11) and is also relatively resistant to inhibition by sCD4 (9, 12). Thus the inhibitory effect on HIV-mediated syncytium formation, which 5A8 has and its Fab lacks (Table 1) (4), could be dependent on a combination of cross-linking of CD4 molecules and steric interference by the Fc portion of the antibody. We do not know how important the particular conformational changes we infer from model systems are for HIV infectivity and syncytium formation. Shedding of gp120, which is blocked by 5A8, may not be necessary for fusion and might indeed abort the fusion reaction prematurely (1, 35). Nor do we know how exposure of previously occult gp4l epitopes influences fusion. However, we suggest that these conformational changes at least partially mimic those happening in the real fusion reaction and that their antagonism by 5A8 accounts for its inhibition of HIV-1 infection. The D3 MAb, Q425 (17), also blocks HIV infectivity at a stage after binding. Its action appears in preliminary experiments to be similar to that of 5A8 but not identical (38a). Furthermore, Q425 and its Fab fragment block sCD4 enhancement of HIV-2 fusion with CD4 cells (8), whereas 5A8 does not (6a). Together, 5A8, Q425, and related MAbs might allow us to dissect further the stages of the HIV-cell fusion reaction.
exposure,
ACKNOWLEDGMENTS
We are grateful to Robin Weiss and David Ho for provision of facilities and for advice. We thank Kathleen Kimball and Renee Shapiro for preparing MAbs and Fab fragments and Dian Olson and Wendy Chen for help in preparing the figures. We are grateful to Susan Zolla-Pazner for MAbs 50-69 and 98-6 and to Franco Celada for MAb F91-55. We also appreciate Paul Clapham's and Thomas Schulz's critical comments on the manuscript and permission from Mitko Dimitrov to quote unpublished results.
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