VIROLOGY

191,732-742

(1992)

Synergistic Interaction between Ligands Binding to the CD4 Binding Site and V3 Domain of Human lmmunodeficiency Virus Type I gpl20 JANE A. McKEATING,*a’

JACKIE CORDELL,t

CHRISTOPHER J. DEAN,t AND PETER BALFE+

*Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 SJB, United Kingdom; tlnstitute of Cancer Research, Section of Immunology, Sutton, Surrey SM2 5NG, United Kingdom; and *Department of Medical Microbiology, University College and Middlesex School of Medicine, Windeyer Building, Cleveland Street, London WlP SDB, United Kingdom Received May 13, 1992; accepted August

19, 1992

We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.139, binding to a conformational epitope of gpl20 involved in CD4 binding, and mAbs binding to the V3 domain of gpl20, can synergistically neutralize human immunodeficiency virus type I (HIV-l). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gpl20 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gpl20 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.139; binding studies demonstrate that these mAbs only bind to virion gpl20 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gpl20 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization. o ISSZ Academic PWSS, IIIC.

INTRODUCTION

(Weiss et al., 1986). Antibodies directed to the third variable (V3) region of gp120 block HIV infection by blocking membrane fusion without affecting the gpl20-CD4 interaction (Linsley et a/., 1988; Skinner et a/., 1988a). V3 antibodies were initially reported to exhibit an isolate-specific or type-specific neutralizing response (Goudsmit et al., 1988a; Palker et al., 1988; Rusche et a/., 1988). However, it has subsequently been reported that the central region of V3 is not as variable among primary HIV-1 isolates as was first thought (LaRosa et al., 1990) and that a number of human and murine antibodies mapping to the “conserved” crown of this region can cross-neutralize multiple HIV-1 isolates with homology in this region (Gorny et a/., 1991; Javaherian et al., 1990; Scott et al., 1990). The more broadly neutralizing response that develops late in infection is not thought to be related to V3 (Goudsmit et a/., 1988b; Profy et al., 1990). Several authors have fractionated HIV+ polyclonal human sera into linear and conformation-dependent components and have suggested that the group-specific neutralization activity is found in the conformation-dependent component (Profy et al., 1990; Steimer et al., 1991). However, the observed neutralization titer of the fractions alone was not equivalent to that of the unfractionated material. Human mAbs binding to the V3 domain and to conformational epitopes involved in gp120CD4 binding, exert greater than additive neutralization when mixed in equimolar combinations (Buchbinder et

The first step in human immunodeficiencyvirus (HIV) infection involves a high-affinity interaction between the outer envelope glycoprotein gp120 and CD4 (Lasky et al., 1987; McDougal et a/., 1986), the cell surface molecule that acts as the primary receptor (Dalgleish eta/., 1984; Klatzman eta/., 1984). This binding is followed by a number of steps involving the transmembrane glycoprotein gp41, CD4 (Camerini and Seed, 1990; Celada et a/., 1990; Healey et al., 1990), and possibly other, as yet undefined, cell surface molecules (Arthos et a/., 1989; Clapham et al., 1991), resulting in the insertion of the amino-terminal gp41 “fusion” domain into the host-cell membrane (Gallaher, 1987). The various regions of the envelope glycoproteins involved in these processes are potential targets for neutralizing antibodies. Sera from many HIV type I (HIV-1)-infected individuals are able to neutralize a broad spectrum of virus isolates, indicating the presence of conserved neutralization epitopes (Berkower et a/., 1989; Robert-Guroff et a/., 1985; Weiss et a/., 1985, 1986). The induction of high titer, broadly cross-reactive, HIV-l-specific neutralizing antibodies by immunization is an important goal in AIDS vaccine development; such broadly neutralizing antibodies have been termed group-specific ’ To whom reprint requests should be addressed. 0042-6822192

$5.00

Copyright 0 1992 by Academkc Press. Inc. All rights of reproduction in any form reserved.

732

SYNERGISTIC

INTERACTION

a/., 1992; Tilley et al., 1992) suggesting that synergism between conformation-dependent and independent IgG fractions may be responsible for the overall neutralization titer exhibited by a polyclonal serum. We have investigated whether other ligands interacting with the CD4 binding site could synergize with antibodies specific for the V3 domain. We show that sCD4 and mAbs binding to conformational-sensitive, but not linear, gpl20 epitopes involved in CD4 binding can synergize with V3 mAbs to neutralize HIV-l (IIIB). We demonstrate an enhanced binding of V3 mAbs to virion gp120 in the presence of sCD4 or mAbs mapping to regions overlapping with the CD4 binding site, and propose that such enhancement is sufficient to explain the observed synergism. Furthermore, two new V3 mAbs are identified which only bind and neutralize IIIB in the presence of sCD4, suggesting that sCD4 binding to virion gpl20 induces a conformational change which increases the affinity of V3 mAbs for their epitopes. MATERIALS Monoclonal

AND METHODS

antibodies

The following gpl20 mAbs (summarized in Table 1) were used for neutralization studies and/or envelopebinding studies: 39.139, specific for a conserved conformational epitope involved in CD4 binding (Cordell et a/., 1991; McKeating et al,, 1992b); 38.1 a, specific for amino acids (aa) 425-441 involved in CD4 binding (Cordell et a/., 1991; McKeating el a/., 1992a); 1 l/65, specific for aa 102-l 21; 41.1 i, specific for a conformational-sensitive epitope within the V3 domain (unpublished data); 1 10.5, specific for aa 3 1O-31 7 in V3 (Kinney-Thomas et al., 1988; McKeating et a/., 1989); 9284, specific for aa 301-312 in V3 (Skinner et a/., 1988b); 0.5@, specific for aa 3 1 l-324 in V3 (Matsushita et a/., 1988); mAbs 8/38c and 8/64b, which map to aa 300-315 in V3; and mAbs 10154, 10/36e, and 11/85b, which map to aa 31 1-321 in V3. Amino acid numbering is according to Myers et a/. (1988). Epitope mapping of the mAbs was evaluated by following the binding of the mAbs to overlapping gpl20 peptides (H. Holmes, MRC ADP) and by the ability of peptides to “in solution” compete for mAb-gpl20 binding (McKeating et al., 1992a). All of the mAbs, with the exception of 1 10.5, 9284, and 0.5p were raised in rats immunized with recombinant gpl20 (HIV-1 IIIB; BHlO clone expressed in CHO cells) emulsified in complete Freunds’ adjuvant via Peyer’s patches. The polyclonal affinity-purified anti-peptide serum D7324 (Aalto Bioreagents, Dublin) was specific for a 15-amino acid peptide (APTKAKRRVVQREKR) from the conserved carboxy1 terminus of gpl20 (Moore et a/., 1990).

733

IN HIV-1 VIRUS

Biotinylation

of mAbs

mAbs were dialyzed against 100 m/l/l sodium hydrogen carbonate buffer, pH 8.4, and then incubated at 500 pg/ml for 16 hr at 4“ with 100 pug/ml of biotin-l\lhydroxysuccinimide ester (Pierce Chemical Co., Rockford, IL). Excess biotin was removed by dialysis against PBS. Biotinylated mAbs were titrated for gpl20 binding by ELISA essentially as described before (Moore et a/., 1990) but using streptavidin-alkaline phosphatase (Novo Nordisk, Cambridge, UK) as the detection system. Concentrations of Biotin-Mab giving half-maximal binding to gpl20 were used for competition assays. Neutralization

assays

HIV-1 IIIB (1 O3TCID,, in a volume of 50 ~1)was incubated with 20 ~1 of RPMI containing agent 1, agent 2, or both agents together for 1 hr at 37”. The virus antibody/sCD4 (Smith Kline Beecham, King of Prussia, PA) or sCD4lgG (Genentech, SF) mixture was then incubated with 100 ~1of c8 166 cells at a concentration of 2 X lo5 cells per milliliter per well in a 96-well plate in triplicate. The amount of supernatant p24 antigen produced in the wells at 3 days postinfection was quantified as described elsewhere (McKeating et al., 199 l), and the reduction in p24 antigen in the presence of the blocking agents was termed the percentage neutralization. Combination

index determination

Neutralization data (Fig. 1a) were transformed using a “median-effect” regression equation (Fig. 1b) (Chou and Talalay, 1984). From the three regression equations for the two agents in isolation and their mixture it is possible to calculate a combination index (Cl) which describes the interaction between the two agents, CL = PWQ)

+ 9KwQ,

where, at a given percentage neutralization, n, D, , D,, and D, are the doses of agent 1 and 2, or of their mixture, m, required to give this amount of neutralization; p and 9 are the relative amounts of agent 1 and 2 in the mixture Co + 9 = 1). By averaging the median effect equations for agents 1 and 2 it is possible to calculate a regression equation which will give a Cl value of 1 (that is, the expected median effect regression equation if the agents act in an additive manner). When this expected regression line lies outside the 95% confidence intervals for the actual median-effect line of the mixture, the corresponding Cl value differs from 1 (additivity) at the 5% (0.05) significance level. This test has been used to assess the significance of the Cl values obtained (Table 2B).

734

Thrombin

MCKEATING

cleavage assays

Soluble and virion gpl20 levels were quantified in culture supernatants from IIIB-infected H9 cells by a combination of S-l 000 gel exclusion chromatography and twin site ELISA (McKeating et al., 1991; Moore et a/., 1990). The total gp120 content of the two viral stocks used in these experiments were 6.4 and 5.5 rig/l 00 ~1, of which 55 and 62% were present as virionbound glycoprotein and the rest as soluble antigen. One hundred microliters of S-l 000 virion and soluble fractions, both containing 450 pg gpl20, were incubated with 10 /II of increasing concentrations of bovine thrombin (Boehringer Mannheim Biochemicals, Germany) in the presence or absence of sCD4 and mAb 39.13g at 37” for 1 hr. The reaction was terminated by the addition of the nonionic detergent Empigen (Calbiochem, Nottingham, UK) to a final concentration of 1%. The extracts were then incubated with plastic-adsorbed D7324 to capture gpl20 onto the solid phase and reacted with a biotinylated preparation of mAb 1 10.5 as described previously (Clements et al., 1990). Thrombin cleavage is detected by a reduction in 1 10.5 binding. Bound 1 10.5 was detected with streptavidinalkaline phosphatase (Dakopatts, High Wycombe, UK) and the AMPAK system (Novo Nordisk, Cambridge, UK) as described previously. Soluble CD4-induced

virion gpl20

shedding

HIV-l IIIB containing culture supernatants were incubated with various concentrations of sCD4 for 2 hr at 37”. To analyze the effect of sCD4 on virion-bound gpl20, virion-bound and soluble gpl20 were quantified by S-l 000 gel exclusion chromatography and twin site ELISA as above. Binding of mAbs and sCD4 to virion and soluble gpl20 Virus (2.7 ng total gpl20) was incubated with mAbs or sCD4 at 0” before passage down a Sephacryl S-l 000 column as previously described (McKeating et a/., 1991; Moore eta/., 1990). Elution was with ice-cold Tris-buffered saline (TBS) and the columns were prechilled on ice. The fraction (600 ~1) containing virions was collected, disrupted with 60 ~1 of 10% Nonidet P-40 (NP-40) plus 109/o fetal calf serum (FCS), and immediately assayed for bound mAb or sCD4. For determination of mAb or sCD4 binding to detergent-solubilized virions, virus was mixed with 19/oNP-40 and incubated for 1 hr at 0” with mAb or sCD4 in a total of 100 ~1 and then diluted with 560 ~1of 1O/oNP-40/FCS to mimic the dilution of virions during gel filtration. The amount of virus in the incubation was adjusted to give a gpl20

ET AL.

concentration that was the same as that in the detergent-solubilized virion fraction eluted from the columns. NP-40 solutions of virion and soluble mAb or sCD4 complexes were captured onto a solid phase by antibody D7324. Bound mAb was detected with alkaline phosphatase-conjugated goat anti-rat IgG/IgM or goat anti-mouse IgG (Seralab, Crawley, UK). Bound sCD4 was detected with a rabbit anti-sCD4 serum (CBL-34) and a sheep anti-rabbit IgG-alkaline phosphatase conjugate (Seralab, Crawley, UK). Note that the anti-rabbit IgG used does not cross-react significantly with rat or mouse IgG, so the presence of gpl20-bound rat or mouse antibodies does not interfere with the assay for gpl20-bound sCD4. Control experiments with 10 pgl ml of mAb in the absence of viral antigen demonstrated that there was no detectable free mAb in the virion fraction.

RESULTS We compared the ability of sCD4, sCD4lgG, and mAbs specific for linear (38.1 a) or conformational-sensitive (39.139) epitopes important in gpl20-CD4 recognition to synergize with a number of V3-specific mAbs (see Table 1, for a summary of mAb epitope recognition). One of the V3 mAbs (41.1 i) binds to a conformational-sensitive epitope within V3 and is unable to bind to synthetic V3 peptides. Recognition of gp120 by 41.1 i is abolished by several mutations within the V3 domain and by thrombin cleavage of the motif GPGRJAF (J.McK., unpublished data). The concentrations of 39.139 and 41.1 i required to neutralize (90% inhibition) 1O3 TCID,, of IIIB were 7.5 and 0.36 pg/ml, respectively. However in combination only 0.65 pg/ml of 39.139 and 0.0125 pg.ml of 41 .li were required to achieve the same level of neutralization (Fig. 1, Table 2A), suggesting that these antibodies in combination synergistically neutralize IIIB. Similar reduced effective doses in mixtures of V3 mAbs and other CD4 binding site ligands are also shown in Table 2A, with the exception of mAb 38.la, binding to a linear component of the CD4 binding site. Synergism may be quantified by the determination of a combination index, where Cl values less than 1 are considered to be synergistic, and those greater than 1 to be antagonistic (Chou and Talalay, 1984). The conformationsensitive mAb, 39.139, was able to exert a synergistic effect in combination with both 1 10.5 and 41.1 i mAbs, giving Cl,, values of 0.013 and 0.002, respectively (Table 2B). In contrast, mAb 38.1 a did not synergise with either of the V3 mAbs tested (Table 2B), giving Cl values that were 50- to 300-fold higher than those ob-

SYNERGISTIC

INTERACTION

IN HIV-1 VIRUS

735

TABLE 1 SUMMARY OF mAbs USED IN THIS STUDY lsotype

Epitope

ML W,,

CD4 binding site CD4 binding site

NMWQEVGKAMYAPPISG

41.li 110.5 9284 0.5p 8/38c 8/64b 10154 10/36e 11/85b

W,, IgG, I& W, k&a bM W, k&a Wm

v3 v3 v3 v3 v3 v3 v3 v3 v3

QRGPGRAF NNTRKRIRIQRG RGPGRAFVTIGKIG NNNTRKRIRIQRGPGR NNNTRKRIRIQRGPGR RGPGRAFVI-IG RGPGRAFVTIG RGPGRAFVTIG

1 l/65

bG,,

Cl

EQMHEDIISLWDQSLKPCVK

mAb 39.139 38.la

Linear peptide

Amino acid residues” 88, 113, 117, 257, 368, 370 425-441 Conformational 310-317 301-312 31 l-324 300-315 300-315 311-321 311-321 311-321 102-121

a The gpl20 amino acid numbering follows that reported by Myers et al. (1988). The epitope mapping of Mabs 1 10.5 (Kinney-Thomas 1988). 9284 (Skinner et al., 1988), and 0.5 p (Matsushita et al., 1988) is as reported in the primary publications.

tained with the 39.139 mAb. sCD4 and sCD4-IgG acted synergistically with both of the V3 mAbs tested to neutralize IIIB (Table 2B). Thus, the ability of agents, binding to discontinuous regions of gpl20 constituting the CD4 binding site, to synergize with antibodies to V3 suggests that ligands may have to bind to gpl20 in a specific manner in order to exert such an effect. Further analysis was performed on various ratios of agents 1 and 2 using a “checkerboard” design (in which titrations of both agents, alone or in combination, were tested). The significance of the observed synergism for the various agent combinations is indicated in Table 2B. The most significant synergism is observed at dilutions of mAbs which alone result in negligible or undetectable neutralization, but in combination exert a significant neutralization. However, the method of analysis devised by Chou and Talalay (1984) cannot utilize data obtained when either of the agents alone fail to neutralize, and thus has considerable disadvantages. We have obtained two V3 mAbs, 8138~ and 8/64b, which do not neutralize IIIB (at final concentrations of 20 pg/ml). However, when these mAbs were incubated with subneutralizing concentrations of sCD4, neutralization was observed (Fig. 2a). Endpoint titrations of mAbs 8/64b and 8138~ in the presence of 0.5 pg/ml sCD4 demonstrated that 50% neutralization was achieved at concentrations of 0.6 and 2.5 /*g/ml, respectively (data not shown). Mab 8/64b also neutralized IIIB in the presence of 39.139, but with reduced efficiency compared with sCD4 (Fig. 2b). In contrast mAb 8/38c did not exhibit significant neutralization in the presence of 39.139 (Fig. 2b). These data suggest that, on viral particles, V3 epitope accessibility is sensi-

et a/.,

tive to changes induced by the binding of ligands to the CD4 binding site. Interaction

between CD4 binding site and V3 domain

A number of experiments were pet-formed to elucidate the mechanism of the observed synergistic neutralization between agents binding to the CD4 binding site and V3 domain. First, we investigated whether mAb occupation of the V3 domain increases the accessibility of the CD4 binding site to mAbs or sCD4. We have previously reported that soluble or recombinant gpl20 (rgpl20) binds sCD4 with a greater affinity than virion-bound or particulate gpl20 (Moore eta/., 1991), suggesting subtle differences between oligomeric virion gpl20 and monomeric rgpl20. We therefore performed mAb and sCD4 binding studies on both virion and soluble gpl20. If occupation of the V3 domain by an antibody increases the accessibility (or epitope exposure) of the CD4 binding site to sCD4 or mAbs, one would expect to observe an increase in the binding of a mAb to this site in the presence of a V3 mAb. We followed the binding of biotinylated 39.13g (at a concentration giving 50% of maximal binding) in the presence of unlabeled 39.139 and 41.1 i, to soluble and virionbound gpl20. Unlabeled 39.139 was found to inhibit the binding of the labeled mAb competitively, whereas 41 .l i had no significant effect, suggesting that the binding of 41 .li to virion (Fig. 3a) and soluble gp120 (data not shown) was independent of 39.139. Comparable results were obtained for the binding of labeled 39.139 to virion gpl20 in the presence of mAb 110.5 (data not shown). We have previously reported that mAbs binding to the CD4 binding site can efficiently

MCKEATING

736

ET AL.

which had been shown by ELISA to saturate this amount of gp120. The amount of sCD4-induced gpl20 shedding was identical irrespective of the presence or absence of 41.1 i or 110.5 (data not shown). Does sCD4 or mAb occupation of the CD4 binding site increase accessibility of V3 to mAbs and thrombin?

n

. . . . ...I

0

.I

0

,“.‘.,I

1

’ .“““I

39,13g/41

.li

. . ..-I

10

100

mAbpglm1

i..l;‘::::. b

1.5

n

a

n

-3 5 tf

1.0

0

l

05

9

0 0 .o l

0

-0.5 0 -1.0 0

.oo 1

.Ol

.l

Diln Inhibitor FIG. 1. Neutralization curves and median-effect plots for mAbs 39.139 and 41.1 i. (a) Neutralization curves for 39.139 (0) and 41.1 i (0) alone and in a 5O:l mixture 39.13g:41 .li (w). (b) Median effect plots of 39.139 (0) and 41 ,l i (0) alone and in a 5O:l mixture 39.13g:41.1 i (m), The Y axis values are obtained as the logarithm of the percentage neutralization (Fa) divided by 100% minus this figure (Fu), i.e., for 50% neutralization Log(Fa/Fu) = 0. The “median effect equations” are the linear regression equations of the three sets of data points.

block rgpl20-sCD4 binding (Cordell et a/., 1991). We therefore followed the ability of 39.139, alone or in combination with 41 .l i, to block rgpl20 binding to sCD4. The presence of 41.1 i did not increase the ability of 39.13g to block the sCD4-gpl20 interaction (data not shown). In agreement with these results, 41.1 i had no effect on the ability of sCD4 to bind to virion (Fig. 3b) or soluble gpl20 (data not shown). sCD4 is thought to neutralize HIV-l infectivity by two mechanisms; competition for cellular CD4, and sCD4induced shedding of the outer envelope glycoprotein gp120 from the transmembrane glycoprotein gp41 (Moore et a/., 1991; Moore et al., 1990). We therefore followed the ability of sCD4 to induce virion gpl20 shedding in the presence of a concentration of 41.1 i

We investigated whether the V3 loop on virion gpl20 was capable of binding increased amounts of V3 mAb in the presence of sCD4. Increasing amounts of sCD4 were incubated with virion or soluble gpl20 at 0” (to prevent virion gpl20 shedding (Moore et a/., 1990)), in the presence of a single concentration of 4 1.1 i (10 pg/ ml), and followed both bound mAb and sCD4. At subsaturating concentrations of sCD4, there was a significant, 2.2-fold, increase in the binding of mAb 41 .l i to viral gpl20; this was not observed with soluble gpl20 (Fig. 4a). Similar results were obtained with a second stock of IIIB virus. The increased OD value for virion gpl20-bound 41 .l i (from 0.24 to 0.52) in the presence of saturating concentrations of sCD4 was close to that obtained for soluble gpl20-bound 41 .li (0.48) suggesting that not all of the epitopes recognized by 4 1.1 i on virion gp120 are available for antibody binding (in contrast to soluble gpl20). This idea was further supported by the inability of mAb 41.1 i to saturate its binding sites on virion gpl20 in the absence of sCD4 at 0” (Fig. 4b). One potential function of the V3 loop is to react with a cell surface protease, resulting in a single cleavage event which may initiate further conformational change(s) in gpl20 and which precedes fusion of the viral and cellular membranes (reviewed in Moore, 1992). We have previously reported that sCD4 can increase the thrombin cleavage of the V3 loop on rgpl20 (Clements et a/., 1990). Thrombin cleavage of gpl20 is followed by the loss of V3 mAb 110.5 recognition, whose epitope includes the thrombin cleavage site (GPGRJAF). Virion-bound gp120 was found to be as equally susceptible to thrombin cleavage as soluble viral gp120 (Fig. 5a) and was enhanced by the presence of sCD4 or mAb 39.139, but not by mAb 38.1 a (Fig. 5b), further suggesting that occupation of the CD4 binding site on particulate gpl20 increases the exposure of the V3 loop to both mAbs and thrombin. We screened a number of V3 mAbs (Table 1) for their binding to virion gpl20 in the presence of sCD4 (10 pglml) at 0”. The mAbs 41 .l i, 1O/54, 10/36e, 1 1/85b, 1 10.5, 9284, and 0.5p demonstrated a 1.4- to 2.2-fold increase in binding to virion gp120 in the presence of sCD4 (Table 3). In contrast, no such increase was observed for their binding to soluble gp120. A control

SYNERGISTIC

INTERACTION

IN HIV-l

VIRUS

737

TABLE 2A NEUTRALIZATIONVALUES FORVARIOUSgpl20

LIGANDS Percentage

Conf

Linear

Conf

Linear

39.139 110.5 39.139 41.li 38.la 110.5

-

-

38.la

41.li sCD4 110.5 sCD4

-

-

41.li

-

sCD4lgG -

’ Concentration(s)

90%

Alone

Mixture

Alone

Mixture

3.30 0.06

0.56 0.004

8.05 0.20

0.97 0.007

3.00 0.13

0.42 0.006

7.50 0.36

0.65 0.0125

9.75 0.05

5.05 0.03

16.85 0.24

9.35 0.18

10.25 0.07

8.05 0.05

17.50 0.25

12.00 0.18

2.00 0.05

0.63 0.003

3.70 0.18

1.05 0.015

2.60 0.06

0.54 0.008

3.40 0.21

1.15 0.08

110.5

-

1.05 0.08

0.17 0.006

2.53 0.22

0.80 0.013

-

41.li

1.20 0.08

0.24 0.01

2.10 0.24

0.85 0.01

sCD4lgG

-

kg/ml)

75%

v3

CD4 BS

neutralization

of ligands resulting in either 75 or 90% neutralization,

mAb, 1 l/65, which is unable to bind to virion gp120, but binds to soluble gp120, showed no detectable binding to virion gpl20 in the presence of sCD4, confirming that sCD4-induced shedding of virion gp120 had not occurred. Two mAbs, 8/38c and 8/64b, which

either alone or in the pairwise combinations

shown.

only neutralize IIIB in the presence of sCD4 (Fig. 2a), bound to virion gpl20 only when sCD4 was present. In addition mAbs, 8/38c and 8/64b, failed to bind to IIIBinfected HeLa-CD4+ cells, whereas mAb 41.1 i bound to these cells, (data not shown). The IIIB-infected HeLa

TABLE 28 Cl VALUES FORVARIOUSgpl20 CD4 BS Linear

38.la 38.la

LIGANDS

v3 Conf

Linear

Conf

%

Cl,,

39.139 39.139

110.5 -

41.li

0.012 0.002

0.012 0.002

0.013 0.002

-

110.5 -

41.li

0.520 0.647

0.591 0.665

0.680 0.702

sCD4 sCD4

110.5 -

41.li

0.024 0.017

0.025 0.007

0.028 0.018