AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 6, 1992 Mary Ann Liebert, Inc., Publishers

Production of Human Monoclonal Antibodies Specific for Conformational and Linear Non-V3 Epitopes of gpl20 S.

KARWOWSKA,1

M.K.

GORNY,1 A. BUCHBINDER,1·3 V. GIANAKAKOS,3 T. FUERST,2 and S. ZOLLA-PAZNER1,3

C.

WILLIAMS,3

ABSTRACT

IgGj human monoclonal antibodies (MAbs) directed against conformational epitopes of the gpl20 envelope protein of HIV-1 were produced, as was a single human MAb to a linear epitope spanning amino acids 487-509 in the C-terminal portion of gpl20. All three conformation-dependent MAbs reacted optimally with recombinant gpl20 (rgpl20) captured on plastic via its carbohydrate moieties with Concanavalin A. These Three

MAbs were able to block the interaction between recombinant CD4 (rCD4) and rgpl20; they were also able to achieve 50% neutralization of HTLV-IIIB and MN strains of HIV-1 in a concentration range of 0.5-12.8 µg/mL. The MAb to the linear determinant is the first reported human MAb specific for the immunodominant portion of gpl20; this MAb was most reactive with rgpl20 when it was coated directly on plastic. It could neither inhibit rCD4-rgpl20 binding nor neutralize either HTLV-IIIB or MN. The binding affinities of the four human MAbs for rgpl20 in solution, reflected by their dissociation constants (Kd), ranged from 0.5 x 10~8 to 7.5 x IO"8 M.

INTRODUCTION INTERACTION OF THE gpl 20 external envelope glycopro¬ tein of HIV-1 with cell surface CD4 is the principal step ' leading to the entry of HIV-1 into CD4+ cells. The structure of the CD4-binding domain (CD4-bd) of gpl20 is under careful scrutiny. It was shown that truncation of large C-terminal fragments, substitution of individual amino acids or deletion of several residues alters CD4-gpl20 binding2-4 and that portions of the second, third, and fourth conserved regions of gpl20 all contribute to the CD4bd.5,6 Antibodies which block the interaction of CD4 and gpl20 have been described. The antibodies derived from animals were stimulated by immunization with peptides or recombinant gpl20;2,7"9 those derived from humans are the result of natural infection.10-13 Studies of these antibodies suggest, as do the structural studies mentioned above, that the CD4bd of gpl 20 is a large, conformation-dependent, discontinuous epitope on the surface of the gp 120 molecule. In order to map this region and to

THE

study the diversity of the human immune response against it, it is optimal to study human monoclonal antibodies (MAbs) specific for this region. Only three such MAbs have been described in the literature.I0-'2·14 However, given the complexity of the CD4bd, several MAbs derived by a variety of screening tech¬ niques are needed to reflect the diversity of this region. For these reasons, several human MAbs reactive with recombi¬ gpl20 from HTLV- (rgpl20HTLV.mB) were produced. Three of these MAbs were specific for the CD4bd; one was specific for a linear determinant in the C-terminal domain of gpl 20. The data from this and other studies suggest that most, if not all, antibodies to the CD4bd in naturally infected hosts are neutralizing since all of these antibodies have been shown to neutralize HIV-1 at microgram levels.11-14 Conversely, anti¬ bodies to the immunodominant region of gpl2015-17 at the carboxy-terminus gpl20, appear lacking in protective function. This is confirmed by results with the first human MAb described to date which is specific for this region and which also lacks nant

neutralizing activity.

'Department of Pathology, New York University Medical Center New York, New York 10016, 2MedImmuneInc, Gaithersburg, MD 20878 and laboratory and Medical Service, New York Veterans Administration Medical Center, New York, NY 10010.

the

1099

1100

KARWOWSKA ET AL.

MATERIAL AND METHODS

Subjects Mononuclear cells were separated from the blood of asymp¬ tomatic and symptomatic HIV seropositive subjects on FicollHypaque as previously described.18 From 9 to 54 x 106 mono¬ nuclear cells were obtained per patient.

Establishment of Epstein-Barr lines and heterohybridomas

virus-transformed cell

The method for producing cell lines synthesizing human MAbs to HIV-1 was previously described.18,19 Briefly, mono¬ nuclear cells were incubated overnight with Epstein-Barr virus. Cyclosporin A (Sandoz, East Hanover, NJ) was incorporated into the medium at 0.5 µg/ml for the first week after in vitro infection of the cells. Cells were cultured for 3-4 weeks in 96-well plates and then screened for Abs in the supernatant as described below. Positive wells were expanded and then fused with the SHM-D33 mouse X human heteromyeloma,20 pro¬ vided by Dr. Nelson Teng, as previously described.19 Fused cells were cultured for 24 h, whereupon 1 x 104 mouse perito¬ neal cells were added as feeder cells and cultures were continued in the presence of hypoxanthine, aminopterin, thymidine, and ouabain. After 2-3 weeks, all cultures were rescreened for specific Ab production and positive cultures were grown in progressively larger culture vessels. Ab-producing hybrids were cloned repeatedly at 100 to 1 cell/well.

Immunoassays To

screen

for Abs to

gpl20,

96-well Immulon-2

plates

(Dynatech, Alexandria, VA) were coated overnight at 4°C with rgpl20HTLV_II1B (Repligen, Cambridge, MA) at a concentration of 0.25 µg/ml in coating buffer, pH 9.6. Recombinant gpl20mB

prepared from silk worm cells infected with recombinant baculovirus containing the gpl20IIIB gene. Culture supernatants were added and incubated for 90 min at 37°C. After washing with PBS-Tween (phosphate-buffered saline, 0.05% Tween-20, pH 7.2), the reaction was detected with alkaline phosphataselabeled goat anti-human IgG antibodies (Zymed, Burlingame, CA) diluted to a concentration of 1 µg/ml. For detection of the ability of anti-gp 120 MAbs to react with rgp 120 bound by various methods, rgp 120HTLV_IIIB or rgpl20SF_2 (generously provided by Drs. N. Haigwood and K. Steimer, Chiron Corp., Emeryville, CA) was bound directly to Immulon-2 plates at concentrations ranging from 50 to 1000 ng/ml. Alternatively, the rgpl20 preparations were indirectly coated by first binding Concanavalin A (Con A, Sigma Chemi¬ cals, St. Louis, MO.) at 200 µg/ml in PBS for 1 h at room temperature. After washing five times with PBS-Tween, one or the other of the rgp 120 preparations was added at the concentra¬ tions noted above. After 4 h at room temperature, the plates were washed and incubated overnight with 15% fetal calf serum (FCS) in RPMI-1640 in order to block unsaturated carbohy¬ drate-binding sites on the Con A. Human MAbs, at 10 µg/ml in PBS-Tween were then added to the plates and the reaction was detected with goat anti-human IgG antibody labeled with alka¬ line phosphatase. PBS-Tween with 1 % bovine serum albumin was used as a negative control. was

To detect the ability of MAbs to block gpl20-CD4 binding, were coated with 1 µg/ml of murine anti-V3HTLV_inB antibodies (American Biotechnology [ABT], Cambridge, MA) for 4 h at room temperature. After washing with TBS-Tween (0.05% TRIS, 0.9% NaCl, 0.05% Tween-20, pH 7.2), the plates were blocked with 1 % BSA in TBS-Tween overnight at 4°C. In a separate tube, human MAbs at various concentrations were mixed in equal volumes with soluble rCD4, an engineered protein made in Chinese hamster ovary (CHO) cells which lacks the transmembrane and intracellular domains of CD4 (Du Pont, Boston, MA) and with rgpl20. The rCD4 was used at a concentration of 500 ng/ml and the rgp 120HTLV.IIIB at a concen¬ tration of 1000 ng/ml. The mixture was incubated for 2 h at 37°C and then added to the coated plates. After 2 h at 37°C, the plates were washed and the presence of rCD4 was detected with alkaline phosphatase-labeled mouse monoclonal anti-CD4 (ABT) at a dilution of 1:2000. This reaction was amplified with the Immuno-select® amplification system (GIBCO, Grand Island, NY). Alternatively, to detect the presence of bound human IgG, alkaline phosphatase-labeled goat anti-human IgG was used. As non-blocking controls, TBS-Tween containing 1% BSA replaced the human MAb. After MAb 450-D was found not to block gpI20-CD4 binding (see below), 450-D was used as a non-blocking control. The percentage of blocking of binding of rCD4 to rgp 120 was calculated by using the formula (^max Ax) I (Amax Amin) x 100, where Ax is absorbance in the presence of a specific blocking antibody at various concen¬ tration, Amax is absorbance in the presence of the nonblocking reagents and Amin is absorbance in the presence of 3.3 µg/ml of MAb that blocks CD4-gpl20 binding. This concentration was within the range of MAbs that gives minimal absorbance (i.e., maximal blocking). For the MAb which appeared not to be

plates

-

-

blocking rgpl20-rCD4 binding (MAb 450-D), the Amin was obtained by averaging the Amin values from the three blocking anti-gp 120 MAbs. Heavy and light chain subclasses were determined by ELISA and IgG was quantitated by methods previously described.IS Except where noted otherwise, radioimmunoprecipitation assays (RIPA) were carried out by the method of Pinter and Honnen21 with 30 µg of HTLV-IIIB (Organon Teknika Corpo¬ ration, Durham, NC) or MN Iysate (Advanced Biotechnologies, Silver Spring, MD) labeled with 125I using the Bolton-Hunter reagent (New England Nuclear, Wilmington, DE). Culture supernatants were incubated with labeled viral Iysate and were further processed for electrophoretic analysis as described.21 When necessary to detect the dependence of antibody reactivity on antigen conformation, labeled Iysate was reduced with 10 mM dithiothreitol (DTT) and then alkylated with 11-mM iodoacetamide. Western blot analyses were performed using kits purchased from Bio-Rad (Richmond, CA). Determination of the dissociation constants (Kd) of human MAbs was performed by an ELISA method according to Friguet et al.22 Briefly, the culture supernatants containing MAbs were tested at concentrations of 0.1-1.5 µg/ml. Recombinant gpl20 preparations in TBS were used at concentrations ranging from 10-6 to IO-9 M. Molar calculations were based on the concen¬ tration of materials provided by the suppliers which were confirmed by our ultraviolet (UV) measurements. Supernatant and rgpl20 were mixed in equal volumes and, after 16 h, the

HUMAN MONOCLONAL ANTIBODIES TO

mixture was added to plates coated with the same species of rgp 120 with which the MAb reacted. The amount of unbound MAb was measured by ELISA using alkaline phosphataseconjugated anti-human IgG and the GIBCO Immuuno-select® amplification system. Data were plotted according to Friguet et al. in order to determine the Kd. The binding region of the conformation-independent antigpl20 human MAb, 450-D, was identified by a radioimmunoprecipitation assay using rgp 120 of HIV-1 expressed by vaccinia virus. The recombinant vaccinia viruses expressing the com¬ plete env gene of HIV-1 isolates HTLV-IIIB (vPE16), RF (vHATD222), and MN (vMN462), have been described previ¬ ously.23,24 In addition, a set of recombinant viruses encoding progressive C-terminal truncated forms of the env protein derived from the IIIB isolate25 was used. These viruses include vPE17 (747aa) and vPE18 (635aa) which encode env proteins with truncated forms of gp41, vPE8 (502aa), vPE20 (393aa), vPE21 (287aa), and vPE22 (204aa) which encode gpl20 in progressively shorter forms. For radioimmunoprecipitation, BSC-1 cells were infected with 20 plaque-forming units (pfu) of recombinant vaccinia virus per cell and metabolically labeled for 10 h with [35S]methionine (Amersham, Arlington Heights, FL). Cells were lysed and proteins were immunoprecipitated as previously described.25 Additionally, the reactivity of antigp 120 human MAb 450-D was analyzed by ELISA on three overlapping peptides spanning residues from 460 to 509 of gpl20 (ABT). Peptides at a concentration of 1 µg/ml were coated on 96-well Immunlon 2 plates, culture supernatants were added and goat anti-human IgG labeled with alkaline phosphatase (Zymed) was used to detect the reaction.

Virus neutralization assay The syncytium-forming infectivity assay of Nara et al.26 was used to measure the neutralizing activity of the MAbs. Briefly, 96-well tissue culture plates were coated with poly-L-lysine, washed, and 5 x 104 exponentially growing CEM-SS cells (gift of P. Nara) were added to each well. Serial twofold dilutions of 50 µ of MAb in RPMI-1640 and 10% heat-inactivated FCS were incubated with 50 µ of viral supernatants (1-2 x 104 syncytium-forming units per ml) for 1 h at room temperature. The cells were exposed to the virus-MAb mixture for 1 h at 37°C. The virus-MAb mixture was then removed, and 100 µ of RPMI/10% FCS was then added. On day 3, 100 µ of medium was added. The syncytia were counted on day 5 or 6. The percent neutralization represents the ratio (multiplied by 100) of the number of syncytia in each well exposed to virus in the presence of antibody over the number of syncytia in control wells exposed to virus in the absence of antibody. All assays were run in duplicate. Note that at no time is human plasma used in this assay and that the antibody does not remain in the culture medium. The dilution at which 50% of the input virus was neutralized was calculated by using the IBM-PC program developed by Chou and Chou.27

RESULTS EBV-transformed peripheral blood mononuclear cells were screened for their ability to produce antibodies

(PBMC)

1101

gpl20

rgpl20IIIB coated directly on ELISA plates. Ap¬ 1-2% of wells showed positive reactivity on this proximately initial screen. Cells from positive wells were then expanded and fused with a heteromyeloma and four heterohybridoma lines were established after cloning. The hybridoma line 559/64-D was generated by fusing Ab-producing cells pooled from two separate patients since each individual patient provided an insufficient number of cells. Each of these four heterohybridomas made MAbs which were reactive with gp 120HTLV.mB and gpl20MN by RIP (Fig. 1 and Table 1), however, only one of the four (450-D) was reactive with gpl20 (from HTLV-IIIB) on Western blot (Table 1). Further studies revealed that reduction and alkylation of viral lysates destroyed the ability of all but one of the MAbs (450-D) to precipitate gpl20 (Fig. 1). This pattern was confirmed by ELISA reactivity with reduced and nonreduced rgp 120 from HTLV-IIIB and SF-2 (data not shown). None of the MAbs were reactive with 19-22-mers of the V3 loops of 8 HIV-1 isolates (data not shown). These data indicate that one MAb, 450-D, is reactive with an epitope available in both native and reduced form, while the three other MAbs, 448-D, 559/64-D, and 588-D, require the native structure of gpl 20 for reactivity. In all cases, specificity would appear to be broad since the four MAbs reacted with the gpl20 of MN, SF-2, and HTLV-IIIB. reactive with

4

5

6

7

8

9

10

·»·tftf 120

*

·

17

FIG. 1. RIP assay of human MAbs with MN Iysate. Reactiv¬ ity of each sample is presented with nonreduced MN Iysate

(lanes 1,3,5,7,9) and reduced and alkylated MN Iysate (lanes 2,4,6,8,10). Lanes: 1 and 2, reactivity of serum specimen from HIV subject; 3 and 4, reactivity of supernatant 450-D; 5 and 6, reactivity of supernatant 448-D; 7 and 8, reactivity of superna¬ tant 559/64-D; 9 and 10, reactivity of supernatant 588-D.

KARWOWSKA ET AL.

1102 Table 1. Characteristics of Human Monoclonal Antibodies Against gp120

Specificity by RIP

Western blot

Cell line

Isotype

(WB)

(WB)

448-D 559/64-D 588-D 450-D

IgGA IgG.K IgG.K IgG,

gpl 20 gpl 20 gpl 20 gpl 20

Negative Negative Negative gpl 20

The IgG subclass of the MAbs was found to be IgG,. Two MAbs (448-D and 450-D) possess lambda chains and two (559/64-D and 588-D) possess kappa chains (Table 1). Determi¬ nation of the dissociation constant (Kd) for each of the MAbs was carried out with rgpl20HTLV.IIIB and rgpl20SF_2. The Kd range "8 M (Table 1). The Kd were consistently was 0.5-7.5 x lower, that is, the affinities were consistently higher, for rgpl20SF_2 despite the fact that these MAbs were isolated on the basis of their reactivity for rgpl20HTLV.,IIB. While this may reflect preferential specificity for SF-2 sequences, it is equally likely that this is a function of the differences between the rgpl20 made in baculovirus (HTLV-IIIB) and that made in Chinese hamster ovary cells (SF-2). At 10 µg/ml, the MAbs could react with as little as 50-100 ng/ml of rgp 120 (Fig. 2). The strength of the reaction was determined, in part, by the nature of the rgpl20 used (baculo¬ virus-derived HTLV-IIIB or CHO-derived SF-2) and by the

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

_"d(xlO-8M)

0.1

0.2

0.3

0.4

0.5

0.6

rgp120 of IIIB (ug/ml)

0.7

HIV-1

HTLV-IIIB

SF-2

IgG Production (µglml) 6 by 1 X 10 cells/ml/24 h

2.9 4.0 2.0 7.5

0.5 1.4 0.6 1.5

17.6 0.9 12.1 11.5

method of coating the gpl20 to ELISA plates (directly, or indirectly with Con A). The differences in reactivity of the MAbs for these various presentations of antigen indicate that the epitope recognized by 448-D, 559/64-D, and 588-D is optimally expressed on rgpl20HTLV.,nB coated indirectly through its carbohydrate residues to Con A (Fig. 2C). MAb 450-D, on the other hand, reacts with an epitope optimally revealed by direct coating of rgpl20SF.2 (Fig. 2b). The inability of these MAbs to react with V3 peptides and their capacity to react with the gpl20 of three HIV-1 strains suggested that these MAbs were specific for shared, or groupspecific epitopes. To determine more precisely the region of gpl20 recognized by these MAbs, their ability to inhibit sCD4rgpl20 binding was tested. Three of the MAbs were able to inhibit the interaction of rCD4 with rgpl20 (Fig. 3). For 50% blocking of the rgpl20HTLV_IIIB and rCD4 interaction, 0.24, 0.10, and 0.11 µg/ml of mAbs 448-D, 559/64-D, and 588-D,

0.8

0.9

1

0

0.8

0.9

1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.7

0.8

0.9

1

rgp120 of SF-2 (ug/ml)

rgp120 of IIIB (ug/ml)

0

of

0.1

0.2

0.3

0.4

0.5

0.6

rgp120 of SF-2 (ug/ml)

FIG. 2. ELISA reactivity of human MAbs with rgpl20 of HTLV-IIIB and SF-2 on plastic coated directly or captured by Con A. Culture supernatants contained human MAbs at a constant concentration of 10 µg/ml. MAbs 448-D ( X ), 450-D ( a ), 559/64-D (* ), and 588-D ( ) were reacted with rgpl20 of HTLV-IIIB (a,c) and SF-2 (b,d) coated directly (a,b) or indirectly via Con A (c,d).

HUMAN MONOCLONAL ANTIBODIES TO

gpl20

1103

10

FIG. 3.

Blocking of the binding between rgp 120 and rCD4 by

15

20

HuMoAbs

(ug/ml)

15 HuMoAbs

(ug/ml)

25

30

human MAbs. MAbs 448-D (X), 450-D ( *), 559/64-D (# ), and 588D ( ) were used at final concentrations ranging from 0.2 to 3.3 µg/ml to block the reaction of rgpl20 and rCD4 used at concentrations of 333 ng/ml and 167 ng/ml,

anti-gpl20

respectively.

respectively, were required. These average 50% blocking endpoints were calculated from experimental data using a linear regression of the median-effect plot.27 These were the same MAbs (448-D, 559/64-D, and 588-D) that required the native gpl20 structure for reactivity (Fig. 1). MAb 450-D was not inhibitory to this interaction. The set of recombinant vaccinia viruses expressing env proteins from multiple HIV-1 isolates (IIIB, RF, and MN), and the nested set of truncated env proteins described in the Material and Methods section was tested for its utility in identifying conserved regions of gpl20 recognized by MAb 450-D. RIPA

confirmed that MAb 450-D could bind the full-length env proteins from isolates III, RF, and MN, as well as the env protein expressed from vPE8 (635aa), indicating it recognized a con¬ served epitope in an extracellular region of the envelope (data not shown). However, further analysis of MAb 450-D revealed that it could not bind to gpl20 with C-terminal truncations suggesting that the binding site resided distal to amino acid residue 393. This was confirmed by the reactivity of MAb 450-D in ELISA with a synthetic peptide spanning residues 487-509. MAb 450-D did not react with peptides spanning residues 460-74 or 474-89 (data not shown). Some, but not all, MAbs to the CD4bd of gpl20 have previously been shown to neutralize HIV.8,1 ',12,14 To determine the capacity of the four MAbs described above to neutralize, their activity in a syncytium-forming infectivity assay was tested. The three MAbs previously found to be conformationdependent and capable of inhibiting CD4-gpl20 interaction were able to neutralize both HTLV-IIIB and MN. Data from representative experiments in which each conformation-depen¬ dent MAb was tested are shown in Figure 4. Experiments with each MAb were repeated two or three times, in duplicate. Average 50% neutralization endpoints were calculated from experimental data using a linear regression of the median-effect plot.27 Fifty percent neutralization endpoints for HTLV-IIIB ranged from 0.5-10.5 µ^ ; MAbs 559/64-D and 588-D were clearly more potent than 448-D. Fifty percent neutralization endpoints for MN ranged from 0.9 to 12.8 µg/ml; all three MAbs were comparable in neutralizing activity against this strain. MAb 450-D, which reacted with denatured gpl20 and

10

20

Inhibitory effect of anti-gp 120 human MAbs on syncytia formation by HIVHTLV.mB and HIVMN. MAbs 448-D

FIG. 4.

Cg), 559/64-D (*) and 588-D ( ) were used at various concentrations. The neutralizing capacity of the MAbs is ex¬ pressed by the surviving virus fraction, in a syncytium-forming infectivity assay using HTLV-IIIB (a) and MN (b). could not block rCD4-gpl20 binding, did not neutralize either of these virus strains.

significantly

DISCUSSION While antibodies to the V3 loop of HIV-1 develop early in the of infection, the titer of anti-V3 antibodies appears to wane during the first year or two of infection.28,29 Antibodies to the CD4bd of gp 120, on the other hand, develop more slowly but apparently persist for a longer period.14 Antibodies to several other epitopes of gpl 20 are also stimu¬ lated by HIV infection.15-17 Some have been associated with neutralizing activity,15,16 but the most immunogenic region of gpl20, the C-terminus, gives rise to Abs which do not neutralize the virus.17 One such antibody in monoclonal form is described above (MAb 450-D). These studies and others suggest that the C-terminus of gpl20 induces a strong but poorly protective antibody response while the V3 region induces a low amount of extremely potent antibodies (30 and Gorny et al., submitted for course

publication).

Since the CD4bd is a common feature of HIV-1, antibodies to this region, which are generally capable of neutralizing viral infectivity, are thought to be relatively broad in their protective capacity.6,8 Monoclonal antibodies to the CD4bd of gpl20 have

1104

previously been defined. Some rodent antibodies which inhibit CD4-gpl20 binding do not neutralize virus infectivity.6,8

However several MAbs (derived from mouse8,9 and from hu¬ man10-12,14 have been characterized which both inhibit CD4gpl20 binding and neutralize HIV infectivity. At least three neutralizing MAbs (two of mouse and one of human origin) directed against the CD4bd do not compete with one another9 suggesting that the CD4bd is large and discontinuous in nature. The specificity and the mechanism by which these antibodies protect against disease need to be understood in order (a) to delineate the human immune response to HIV, (b) to formulate vaccines that can induce these antibodies, and (c) to use these antibodies for passive immunotherapy. The three MAbs to the CD4bd described above were identi¬ fied using a screening technique designed to identify MAbs which were highly cross-reactive. Thus, initial screens for reactivity were performed with gpl20HTLV_II1B. Since the prev¬ alence of this strain in North American patients is extremely small,31,32 it was anticipated that antibodies derived from the majority of HIV-infected subjects and reactive with gpl201IIB would represent antibodies to shared epitopes. This hypothesis would appear to be supported since all three MAbs react comparably with HTLV-IIIB, MN, and SF-2 strains of HIV-1 (Table 1 and Fig. 1) and neutralize all three strains of the virus (Fig. 4 and unpublished data). The anti-CD4bd MAbs described above are distinguished by their ability to react with nanogram quantities of rgpl20 in ELISA and by high affinity for rgp 120 (Table 1., Fig. 2). They mediate 50% blocking of the gpl20-CD4 interaction in a competitive assay at concentrations of 0.1-0.25 µg/ml. This is comparable to the blocking activity of the anti-CD4bd described by Ho et al.14 under noncompetitive conditions. Moreover, all three MAbs mediate 50% virus neutralization at 0.5-12.8 µg/ml. This activity is comparable to that described by Ho et al.14 and Tilley et al.12 for human MAbs to the CD4bd, but is considerably more potent than the activity described by Posner et al." for another human MAb to the CD4bd. Like the previously described human MAbs to the CD4-binding domain, the MAbs described here are all conformation dependent. The relative neutralizing efficiency of antibodies to the V3 loop and to the CD4bd can begin to be clarified with the existence of several human MAbs to both regions. Thus, human MAbs to the V3 loop are active at nanogram levels when tested against the virus strain for which they are specific. For example, using the syncytium-forming infectivity assay, human MAbs to the V3MN region were shown to induce 50% neutralization of HIV-1MN at concentrations as low as 0.05 µg/ml.19,33 In the data shown above, the anti-CD4bd MAbs displayed 50% neu¬ tralizing doses for the MN and IIIB virus strains of 0.9-12.8 and 0.5-10.5 µg/ml, respectively. The difference in potency be¬ tween anti-V3 and anti-CD4bd MAbs for MN strain is as much as 250-fold. Moreover, since these two categories are primarily composed of IgG, MAbs and their affinities are comparable ( 19 and Table 1), it would appear that these parameters cannot account for their different levels of activity. The mechanisms by which these two categories of antibodies neutralize virus infectivity may provide a more cogent explana¬ tion of their different efficacies. Thus, it appears that anti-V3 antibodies prevent infection at a postbinding step since they do not inhibit binding of the virion to the cell, while they are

KARWOWSKA ET AL.

neutralizing after exposure of cells to virus.34 It has been suggested that these MAbs may block infectivity by preventing the cleavage of the V3 loop by a cell-associated protease.35

Anti-CD4bd antibodies, on the other hand, do appear to prevent the binding of the virus to cell surface CD4.34 Thus, this latter category of antibodies are acting, not as inhibitors of a reaction, but as competitors for the CD4-gpl20 binding reaction. Indeed, the anti-CD4-bd MAbs described here, with Kd of 0.5-7.5 x IO-8 M, must compete with a CD4-gpl20 reaction for which the Kd value has previously been reported in the nanomolar range.2,36,37 Given these conditions and mecha¬ nisms, the greater efficacy of the anti-V3 antibodies can be understood. The anti-CD4-binding domain antibodies may, nevertheless, prove to be important in protecting against infection and in have the slowing disease progression. Thus, these antibodies ' advantage of recognizing many (but not all1 -13·14) strains of the virus, whereas the anti-V3 antibodies, while often cross-reac¬ tive,38-40 can be considerably less potent against heterologous strains or variants. Finally, the anti-CD4bd antibodies have been shown recently to act in synergy with anti-V3 antibodies, increasing the potency of both and extending the effective biological activity of the latter.33 Thus, it is likely that active immunization will require that antibodies to both regions be stimulated and passive immunotherapy may require the use of cocktails that contain antibodies to at least both of these regions.

ACKNOWLEDGMENT This project was supported in part by the Center for AIDS Research (NIH award AI 27742) and by research funds from the NIH (AI 72658 and AI 32424) and the Department of Veterans Affairs.

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7. Dowbenko D, Nakamura G, Fennie C, Skimasaki C, Riddle L, Harris R, Gregory T, and Lasky L: Epitope mapping of the human immunodeficiency virus type 1 gpl20 with monoclonal antibodies. J Virol 1988;62:4703-4711. 8. Sun N, Ho DD, Sun CRY, Liou R-S, Gordon W, Fung MSC, Li X-L, Ting RC, Lee T-H, Chang NT, and Chang T-W: Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gpl20. J Virol 1989;63:3579-3585. 9. Ho DD, Fung MSC, Cao Y, Li XL, Sun C, Chang TW, and Sun : Another discontinuous epitope on glycoprotein gp 120 that is impor¬ tant in human immunodeficiency virus type 1 neutralization is identified by a monoclonal antibody. Proc Nati Acad Sci (USA)

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