AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 5, 1990 Mary Ann Liebert, Inc., Publishers

Identification of Conserved and Variant Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) gpl20 by Human Monoclonal Antibodies Produced by EBV-Transformed Cell Lines JAMES E. ROBINSON, DEBRA HOLTON, SUSAN PACHECO-MORELL, JAMES LIU, and HAL McMURDO

ABSTRACT

Using Epstein-Barr virus (EBV) transformation of B cells isolated from peripheral blood of type l-(HIV-l) infected subjects, we have four human monoclonal antibodies (HMAbs) that bind to HIV-1 gpl20, as IgGj produced determined by Western blot analysis. Two of these HMAbs, designated N70-1.5e and N70-2.3a, react with epitopes of gpl20 expressed by all strains tested thus far, and therefore, appear to identify conserved epitopes. The other two HMAbs, K24-3b and N70-1.9b, identify variant epitopes; K24-3b binds to an epitope which is absent from two strains but heterogeneously expressed in eight other strains; N70-1.9b binds to an epitope that is found in relatively few strains. We also describe a novel immunoassay in which viral glycoproteins, produced by HIV-1-infected cells grown in serum-free medium, are affinity immobilized in Concanavalin A-coated wells of enzyme-linked immunosorbent assay (ELISA) plates. This method greatly facilitates the preparation of solid-phase HIV envelope glycoproteins from multiple virus strains and screening immunoassays based on this method are highly sensitive and effective in detecting antibodies to gp!20. two asymptomatic human immunodeficiency virus

INTRODUCTION

The(HIV-1)

gpl20, the extracellular glycoprotein of human immunodeficiency virus type 1 is highly variable among different strains, including sequential isolates from the same patient.1_4 The greatest variability has been demonstrated to be nonrandomly arranged in several hypervariable regions which are flanked by more conserved regions.3-4 The predicted structure of gpl20 suggests that variable regions form potential antigenic sites.3-4 If hypervariable domains of gpl20 elicit the production of gene encoding

Departments of Pediatrics and Microbiology, Immunology

Medicine, New Orleans, Louisiana 70112.

567

and

Parasitology,

Louisiana State

University

School of

ROBINSON ET AL.

neutralizing antibodies,

it is possible these antibodies could select for the emergence of neutralizationresistant variant viruses that evade the host immune system, thereby contributing to HIV-1 disease pro-

gression.

Much of our present knowledge about which domains of gpl20 are immunogenic comes from studies of antibodies raised in laboratory animals. It was first observed that a variety of animals immunized with purified or recombinant gpl 20 or gpl 60 developed strain-specific neutralizing antibodies.57 Subsequently, polyclonal animal antisera raised to recombinant or synthetic peptides representing the third hypervariable domain (V3) of gpl20, covering amino acid residues 307-330, were found to manifest similar strain-specific neutralizing activity, thus identifying the V3 domain as a major target of neutralization, at least in laboratory animals.8-9 In addition, several type-specific neutralizing murine monoclonal antibodies have been produced that bind to epitopes within the V3 domain,10"13 allowing precise mapping of these epitopes. Murine monoclonal antibodies have also been generated to epitopes associated with the putative CD4-binding region near the COOH terminal of gpl20,14"15 but the extent to which this region is also involved in neutralization has not yet been conclusively established. Although the peptide sequence in this region is relatively well conserved, Lasky et al.14 detected minor sequence differences in several virus strains, raising the possibility that some antigenic variation may also occur within the CD4-binding domain. Knowledge regarding which epitopes of gp 120 stimulate humoral responses in chronically infected patients is limited. In contrast to the type-specific neutralizing activity of animal antisera, sera of HIV-infected patients generally contain more broadly reactive, group-specific neutralizing antibodies.15"17 Nevertheless, several groups of investigators have observed that some patient sera manifest distinct patterns of strain-restricted neutralizing activity when tested against a variety of strains.5-61819 Moreover, patient antibodies affinity purified to gpl20 from one virus strain show type-specific neutralizing activity against that strain.6 These observations suggest that the multiplicity of antibodies commonly detected in HI V-1 -positive patient sera may mask important antibody responses to variable neutralization epitopes. Epitopes recognized by immunized animals may differ from those that stimulate antibody responses in naturally infected patients. In order to more precisely identify and characterize both conserved and variant determinants of gpl20 that are recognized by the human immune system, we have sought to produce human monoclonal antibodies (HMAbs) that represent B-cell responses to antigens in gpl20 that have immunized chronically infected patients. Herein, we report the characteristics of four IgG HMAbs with specificity for gpl20 which were produced by Epstein-Barr virus- (EBV) transformed B cells derived from peripheral blood lymphocytes of two asymptomatic HIV-1-infected subjects. Two HMAbs bind to relatively well-conserved epitopes, whereas the other two HMAbs identify variant epitopes.

MATERIALS AND METHODS

Epstein-Barr virus transformation Peripheral blood mononuclear cells (PBMC) from two adult HIV-1-seropositive male subjects were isolated on Ficoll-hypaque gradients. PBMC were depleted of CD3-positive T cells using an indirect panning technique20 in which cells reacting with the OKT3 monoclonal antibody were absorbed to petri dishes coated with F(ab)2 antibodies to mouse IgG. Nonadherent cells, enriched in B cells, were inoculated with the B95-8 strain of EBV21 and plated at 103 or 104 cells per well in 96-well tissue culture plated with irradiated human umbilical cord blood lymphocytes (105 cell/well) as feeder cells. Cultures were maintained in RPMI 1640 containing 5% fetal calf serum (FCS) and 1% Nutridoma-Hu (Boehringer-Mannheim), a serum substitute of low protein content. ELISA Two methods

were

used to immobilize HIV-1

(ELISA) plates for antibody screening. In

one

antigens in wells of enzyme-linked immunosorbent assay system, used only in the first transformation experiment,

HIV-1 -infected H9 cells were immobilized in Concanavalin A- (Con A) coated assay wells and then fixed with

568

HUMAN MONOCLONAL ANTIBODIES TO HIV-1

gpl20

1:1 acetone-methanol. Immobilized, fixed uninfected H9 cells were used as control antigen. The wells were blocked with RPMI-10% FCS for 1 h. Fluids from 96-well cultures were transferred to wells in the assay plates. After 1 h, wells were washed with phosphate-buffered saline (PBS) containing 0.1% Tritôn-X 100 (PBS-Tx) and then reacted with peroxidase-conjugated antibody to human IgG (Protos Labs). Color was developed with 100 p.1 tetramethylbenzidine (TMB)-H202 as substrate. The reaction was stopped by the addition of H2S04 and color was read as optical density (OD) at 450 nm in a Titertek Multiskan ELISA reader. The other ELISA method was a novel immunoassay based on the observation that HIV envelope glycoproteins bind via their carbohydrate moieties to Con A.22 Wells of Immulon-2 assay plates (Dynatech) were coated with 200 p.g/ml Con A in PBS and then incubated with 100 p.1 of detergent-disrupted supernatant fluids from HIV-1 producer cell lines grown for 2-3 days in serum-free RPMI supplemented with 1% Nutridoma-Hu. In the absence of serum components, disrupted viral glycoproteins present in such culture fluids bind to Con A in amounts sufficient to function as solid-phase antigens in a highly sensitive ELISA. Unreacted Con A-binding sites were blocked with RPMI-10% FCS for 1 h. Control antigens were similarly prepared from culture fluids of uninfected MT4 cells. Transformed B-cell culture fluids were transferred to both antigen-coated and control wells of assay plates which were incubated at room temperature for 1 h. Binding of antibodies were measured as in the first assay. This ELISA was also used in later experiments to test the reactivity of HMAbs with glycoproteins from different virus strains.

HIV strains Nine HIV-1 strains were used in these studies. Strains C39, J62, SA90, SA96, and L86 were isolated in our

laboratory from mitogen-activated T cells of five asymptomatic HIV-1 -infected subjects by cocultivation with activated normal T cells in medium supplemented with interleukin-2 (IL-2). Strain SA3 was similarly isolated in our laboratory from a patient with AIDS; strain HiTi23 was obtained from Robert Garry, Tulane Medical School; strain K3 was obtained from S.R.S. Rangan of the Tulane Delta Primate Center; HTLV-IIIB,24 the prototype HIV-1 strain, was obtained from American Type Culture Collection; HTLV-IIIIMN,25-26 as well as glycosylated recombinant gpl20 from HIV-1SF2,27 were obtained from the AIDS Research and Reference Reagent Program. Strains C39, J62, SA96, and SA90 were grown in MT4 cells;28 HTLV-IIIB, HTLV-IIIMN, SA3, HiTi, and K3 were grown in H9 cells. Strain L86, isolated from the B-cell donor of one monoclonal antibody (K24-3b), did not replicate in continuous T-cell lines and was propagated in mitogen-activated cord

blood T cells in medium containing 100 units/ml recombinant IL-2. To prepare antigens for ConA immobilization, cells infected with each virus strain were grown for 2-3 days in serum-free medium RPMI supplemented with 1% Nutridoma-Hu. Clarified fluids were treated with 1% Triton-X and stored in aliquots at-20°C until use.

Western blot and dot blot assays Extracts of 1-2 x 107 HIV-1-infected cells prepared by solubilizing cells for 30 min in 1% Triton-X followed by removal of insoluble material by centrifugation in a microcentrifuge. Samples were mixed 1:1 with sodium dodecyl sulfate (SDS) sample buffer without reducing agents and heated for 5 min at 95°C. Cell lysates of uninfected H9 and MT4 cells were similarly prepared. Samples were fractionated by electrophoresis in 7.5% sodium dodecyl sulfate-polyacrylamide gells (SDS-PAGE) in a BioRad minigel apparatus. Proteins were then electrophoretically transferred to nitrocellulose membranes. Western blot strips were incubated with blocking buffer ( 1 % bovine serum albumin, 0.5% Tween-20, in 0.5 M NaCl, 10 mM Tris, pH 8), reacted first with each antibody preparation and then with alkaline phosphatase-conjugated antibodies to human or sheep IgG, as appropriate. Colored bands were developed using nitroblue tetrazolium and 5-bromo4-chloro-3-indolyl-phosphate (NBT-BCIP) as substrate. A sheep antiserum to gpl20 of HTLV-IIIB, obtained from the AIDS Research and Reference Reagent Program, was used as positive control in detecting gp 120/160. For dot blot assays, strips of nitrocellulose were dotted with 1 p.1 of baculovirus-produced, recombinant LAV gpl 20 at 100 p-g/ml (kindly provided by J .L. Raina, American BioTechnologies, Inc., Cambridge, MA) and J62 envelope glycoproteins, which were partially purified from detergent-treated serum-free culture 569

ROBINSON ET AL. medium by lentil lectin affinity chromatography22 and concentrated to 10 p,g/ml. Recombinant gpl20 was also dotted after being heated for 5 min at 95°C in the presence or absence 2-mercaptoethanol. Antibody assays on dot blot strips were performed as for Western blots, except a goat antiserum to gpl60 of HTLV-IIIB29 was used as a positive control.

RESULTS Isolation

of antibody-producing B-cell lines

In the first transformation experiment, EBV-exposed, T-cell-depleted PBMC from a HIV-1-infected donor plated at 103 cells/well in 96-well culture plates with irradiated HUCL feeder cells. Only about 50% of the cultures were transformed after 4 to 5 weeks of culture. Culture fluids were screened by ELISA for IgG antibodies reacting with fixed, immobilized HIV-infected H9 cells, but not with uninfected H9. One transformed culture, designated K24-3b, produced an antibody that appeared on screening to react specifically with an HIV antigen. This culture was a stable producer of antibody, which on further testing reacted by indirect immunofluorescence with both fixed and unfixed HIV-1-infected cells, but not with uninfected cells. Multiple subcultures of K24-3b cells were established at low cell density and all subcultures continued to produce antibody. We were unable to definitively clone K24-3b and, after about 8 months, all subcultures ceased to grow; but by that time we accumulated about one liter of antibody-containing culture medium. Because the original cells were plated at a relatively low cell density and the incidence of transformation was less than 50%, it is likely that the K24.3b cell line was established as a clone. In the second experiment, EBV-exposed T-cell-depleted PBMC from another HIV-positive subject were seeded at 104 cells/well with irradiated HUCL in two 96-well plates. Transformation occurred in 100% of the wells. Culture fluids were screened by ELISA for IgG antibodies reacting with Con A-immobilized viral glycoproteins derived from the J62 strain of HIV-1 grown in MT4 cells in serum-free medium. Ten transformed cultures initially produced IgG antibodies reacting with J62 glycoproteins but not with control antigen. Seven cultures produced antibodies for less than two months. Three cell lines, designated N70-2.3a, N70-1.5e, and N70-1.9b, respectively, were stable antibody producers and after passage through a series of low cell density subcultures these antibody-producing cells were cloned at 10 cells/well. Clones of each line have been stable with respect to growth and antibody production for over 10 months. The IgG subclass and light chain type of each antibody was determined by reactivity with murine monoclonal antibodies to the four heavy chain subclasses (Behring Diagnostics) or polyclonal goat antibodies to lambda and kappa light chains in a sandwich ELISA. All four HMAbs are of the IgG, subclass; K24-3b, N70-1.5e, and N70-1.9b contain kappa light chains and N70-2.3a contains lambda light chains. Culture supernatants harvested weekly from the cloned cell lines contained 1-10 p.g IgG/ml as determined by quantitative IgG capture ELISA. were

Characterization

of HMAb specificity by

Western blot and dot blot assays

Figure 1 shows the reactivity of the four HMAbs on Western blots of antigens of two HIV-1 strains, HTLV-IIIB and J62. On blots of HTLV-IIIB (Panel A) three HMAbs (K24-3B, N70-2.3a, and N70-1.5e) reacted strongly with a prominent band of approximately 120 kD and with a less intense band of 160 kD. N70-1.9b did not react with HTLV-IIIB. On blots prepared from strain J62 (Panel B) all four HMAbs showed identical binding to a prominent band at 160 kD below which was a diffuse band extending to approximately 120 kD; this pattern is characteristic for this strain. The staining patterns obtained with a polyclonal sheep antibody to gp 120 on blots of both strains were identical to that observed with the monoclonals (lane 5 in panels A and B). The HMAbs did not react with blots of uninfected MT4 or H9 cells (not shown). While these results appeared to indicate that these four HMAbs react with gpl20 and its uncleaved cellular precursor, gpl60, Zolla-Pazner et al.30 recently presented evidence that bands identified as gp 160/120 in some commercially available HIV-1 Western blot strips are multimers of gp41. To further confirm that the four HMAbs are indeed specific for gpl20, we tested their binding on dot blots of recombinant LAV gpl20 570

HUMAN MONOCLONAL ANTIBODIES TO HIV-1

gpl20

A. IIIB 12 3 4 5

I 1 '

160120-

FIG. 1. Western blot analysis of HMAb reactivity with two strains of HIV-1. (Panel A) HTLV-IIIB; (Panel B) HIV-1 strain J62. The reactivity of four HMAbs with strips in both panels is as follows: Lane 1, K24-3b, Lane 2, N70-2.3a, Lane 3, N70-1.5e, Lane 4, N70-1.9b, Lane 5, reactivity of sheep anti-HTLV-IIIB gpl20.

lectin-purified J62 glycoproteins. As shown in Figure 2, three of the four HMAbs (K24-3b, N70-2.3a, and N70-1.5e) reacted strongly with recombinant gp 120. N70-1.9b did not bind to LAV gp 120 but and lentil

did bind to J62 antigen. The amount of J62 antigen dotted was about tenfold less than the recombinant antigen, explaining the weaker staining observed with this antigen. These results, therefore, indicate that the bands of 120 and 160 kD observed on our Western blots indeed represent gp 120/160.

2

gpl20J62 •

rgpl20LAV

#

rgpl20 Reduced rgp 120 Heated

FIG. 2. Reactivity of four HMAbs with dot blots of purified gpl 20 of strain 162, nonreduced recombinant LAV gp 120, reduced recombinant LAV gpl20, and nonreduced, heated LAV gpl20.

571

ROBINSON ET AL.

preliminary Western blot studies, neither K24-3b nor N70-2.3a reacted with blots prepared from cell lysates heated in sample buffer containing 2-mercaptoethanol (not shown), suggesting that the epitopes identified were sensitive to reduction. To further test the effect of reduction on these epitopes, the antibodies were tested on dot blots of recombinant gpl20 LAV heated at 95°C in the presence or absence of 2-mercaptoethanol. The results shown in Figure 2 demonstrate that K24-3b and N70-1.5e did not bind to reduced antigen and binding of N70-2. 3a to reduced antigen was significantly diminished, while heating alone only slightly diminished antigenic activity. As N70-1.9b did not bind to LAV gpl20, the effect of reduction on its epitope was not determined in this experiment. We have subsequently tested N70-1,9b on dot blots of reduced and nonreduced J62 glycoproteins and observed no reactivity with reduced antigen. Thus, all four HMAbs identify reduction sensitive epitopes. In

Analysis of strain specificity of HMAbs by ELISA The four HMAbs were tested by ELISA for reactivity with ConA-immobilized viral glycoproteins from different HIV-1 strains. In one experiment (Fig. 3), culture fluids of K24-3b and N70-2.3a, and a HIV-1-positive control serum (H72) were tested on a panel of nine different strains, which included L86, the strain isolated from the B-cell donor of K24-3b. N70-2.3a reacted with all nine strains. Although some differences in binding of this antibody on the panel were observed, generally parallel differences were observed with the positive control serum. Thus it is likely that the binding levels of both N70-2.3a and the H72 serum provide a relative measure of the amounts of gpl20 immobilized from each strain. We have no explanation for the much weaker reactivity of N70-2.3a with the L86 strain compared with the positive serum. However, L86 was the only strain growth in IL-2-dependent primary T cells, which release less virus than continuous cell lines. Possibly more gp41 than gpl20 was immobilized in the L86 virus preparation, and antibodies to gp41 account for the greater serum reactivity. By comparison to N70-2.3, the K24-3b monoclonal showed remarkable variability in reactivity with these viruses. This antibody reacted with six of the nine strains but did not bind to strains SA3 or K3, and showed minimal binding to strain SA96. Whereas the reactivity of both N70-2.3a and K24-3b to strains L86 and J62 were very nearly the same, the binding of K24-3b to strains SA90 and C39 was much less than N70-2.3a, the difference being greatest with SA90. These observations have been reproducible in assays performed with different batches of antigens. Smaller differences in binding of these two antibodies was also apparent with

C39

IB

L86

SA90

SA96

K3

SA3

FIG. 3. ELISA reactivity of K24-3b and N70-2.3a HMAbs with ConA-immobilized gpl 20 from nine strains of HIV-1. Results shown as mean OD of triplicate determinations; standard deviation bars are shown. H72, code designation of an HIV-1 antibody-positive control serum.

572

HUMAN MONOCLONAL ANTIBODIES TO HIV-1

N70-2.3a

N70-1.9b

gpl20

N70-1.5e

FIG. 4. ELISA reactivity of N70-2.3a, N70-1.5e, N70-1.9b HMAbs with ConA-immobilizedgpl20 from 8 strains of HIV-1. Results shown are single determinations.

strains HiTi and HTLV-III. These differences in reactivity were not due to differences in antibody concentration, since the concentration of each supernatant antibody used in these assays were sufficient to saturate the available antigenic sites on immobilized glycoproteins; that is, the OD readings observed with serial dilutions of both antibodies tested against the J62 isolate began to decrease significantly only at dilutions of 1:16 or 1:32 (data not shown). We interpret these data as indicating that N70-2.3a identifies a conserved epitope, while K24-3b binds to a variant epitope which is heterogeneously expressed in this panel of virus strains. Figure 4 illustrates the results of a similar experiment comparing the reactivities of N70-1.5e and N70-1.9b with ConA-immobilized glycoproteins derived from 8 strains. In this experiment, N70-2.3a served as a positive control. Whereas, both N70-1.5e and N70-2.3a reacted strongly with all 8 strains, N70-1.9b reacted only with J62, the strain that was used in the screening the original B-cell cultures for antibody production. The results indicate that N70-1.5e, like N70-2.3a, reacts with an epitope shared by all strains tested thus far, while N70-1.9b reacts with a highly strain-restricted epitope. However, in a subsequent experiment, we found that N70-1.9b, as well as the other three HMAbs, reacted strongly by ELISA with ConA-immobilized glycoproteins from two other strains, HTLV-IIIMN, and HIV-1SF2. These findings (Table 1) indicate that the N70-1.9b-defined epitope is not as highly strain restricted as the results shown in Figure 5 initially suggested.

Strain

specificity of HMAbs by

Western blot

analysis

The strain specificity of two of the four HMAbs, K24-3b and N70-2.3a, were also tested on Western blots prepared from the above panel of HIV-1 strains. The results, shown in Figure 5, are in agreement with results obtained by ELISA. N70-2.3a reacted with gpl20/160 of all 8 strains. K24-3b reacted with gpl20/160 of the same strains it identified by ELISA; it failed to react with SA3 and K3, and its weak reactivity with SA96 was below the sensitivity of photography. Although N70-1.5e and 1.9b have not been similarly tested by Western blot on all of the viruses, the strain-restricted reactivity of N70-1.9 observed by ELISA is corroborated by its failure to react with gp 120/160 of HTLV-IIIB by Western blot or with recombinant LAV gp 120 in the dot blot assay. 573

ROBINSON ET AL. Table 1. Reactivity of Four HMAbs with Con A-lMMOBILIZED gpl20 OF THREE HIV-1 STRAINS

Optical density with indicated strain* HTLV-IIlMNb rgpl20/HIV-]SF-2c

HMAb K24-3b N70-2.3a N70-1.5e N70-1.9b

0.681 1.149 1.648 1.307

1.870 2.018 1.924 1.710

J62h 1.882 2.007 1.781 1.397

"Background reactivity of HMAbs with blocked Con A-coated antigen was < 0.100. bHTLV-IIlMN and J62 viruses grown in serum-free medium as in Figures 4 and 5. cRecombinant glycosylated gpl20 from HIV-lsF2 produced in chínese hamster ovary cells; gpl20 incubated at 1 /Jg/ml 'n Con Awells without

coated wells.

A

C39

11 IB

HiTi

J62

SA90

SA96

K3

SA3

C39

II IB

HiTi

J62

SA90

SA96

K3

SA3

160120-

B 160 120-

-

FIG. 5.

.......

Reactivity of K24-3b and N70-2.3a HMAbs with Western blots prepared from eight independent HIV-1 strains

(Panel A) Reactivity of K24-3b; (Panel B) Reactivity of N70-2.3a. The different strains are indicated by code numbers at the top of each blot lane; IIIB refers to HTLV-IIIB. 574

HUMAN MONOCLONAL ANTIBODIES TO HIV-1

gpl20

DISCUSSION We report the production of four IgG HMAbs with specificity for gp 120/160 of HIV-1 B-cell line's derived by EBV transformation of peripheral blood B cells obtained from two asymptomatic HI V-1-infected patients. Two HMAbs (N70-2.3a and N70-1.5e) bind to epitopes shared by all strains tested thus far, and therefore, tentatively identify conserved epitopes. The other two HMAbs bind to variant epitopes. The K24-3b HMAbs identifies an epitope that is lacking in two virus strains and heterogeneously expressed in nine other strains, while N70-1.9b identifies an epitope expressed in only 3 of 10 strains tested. This epitope would also be expected to be expressed in the virus strain infecting the N70 donor, but we were unsuccessful in isolating this strain. Together these results demonstrate the feasibility of using HMAb production by EBV-transformed cells to probe human antibody responses to both variant and conserved epitopes of gpl20 that are immunogenic in chronically infected hosts. Other HMAbs to HIV-1 proteins have been reported in several publications, but these antibodies have reacted either with gag proteins or gp41.31"35 We are also aware that several laboratories have produced HMAbs to gpl20, but details on these antibodies have not yet been published. Although several of these HMAbs were produced by hybridoma formation,32 a majority were produced by EBV transformation alone, or in combination with secondary hybridoma formation. We, and others,34-36 have observed that peripheral blood B cells from HIV-1-infected subjects vary greatly in their susceptibility to EBV transformation. In general, B cells from patients with severely impaired immune function and relatively low CD4 cell counts34 are the most resistant to transformation, whereas B cells from asymptomatic patients with relatively high CD4 cell counts tend to transform readily. However, we have also observed that transformation rates are variable even within the population of apparently healthy asymptomatic patients (unpublished) and not all attempts to produce HMAbs from this group have been successful. Presently there seems to be no way to predict which subjects are most suitable as B-cell donors for HMAb production. Results of several collaborative efforts to further characterize these monoclonals will be presented elsewhere, but can be summarized as follows. Purified N70-1.9b reacts with synthetic peptides covering the third hypervariable domain (V3) of the HTLV-IIIMN virus strain and neutralizes strains that share V3 sequences with HTLV-IIIMN at endpoint antibody concentrations of 0.5 p.g/ml (Scott C et al., submitted). Purified N70-1.5e neutralizes a broad spectrum of HIV-1 strain at antibody concentrations as low as 1 p.g/ml and binding of this antibody to gpl20 is inhibited by rCD4, suggesting that this antibody inhibits virus-receptor binding (Ho DD, manuscript in preparation). The potent neutralizing activity of N70-1.5e against HTLV-IIIB has been confirmed in another laboratory (Tekeda A et al., manuscript in preparation). Although the binding sites of K24-3b and N70-2.3a have not been identified, the latter antibody mediates antibody-dependent enhancement ( ADE) of virus entry via Fc receptors on U937 cells ,37 but shows little or no neutralizing activity. By contrast, N70-1.5e neutralizes but does not enhance HTLV-IIIB infectivity in the same system (Tekeda A et al., manuscript in preparation). These results indicate that at least one epitope involved in neutralization is distinct from at least one epitope involved in ADE. However, whether neutralizing epitopes are always distinct from enhancing epitopes remains to be determined. Studies comparing the neutralizing and virus-enhancing activity of other HMAbs to gpl20 should provide answers to this question. None of the four HMAbs reacted with reduced gpl 20 by either Western blot or dot blot assay. Since these antibodies were derived from asymptomatic donors, these results are consistent with the earlier observations of Chou et al.38 that antibodies in the sera of patients at early stages of HIV-1 infection react with nonreduced, but not with reduced gpl20 on Western blots, while antibodies reacting with reduced gpl 20 are detected only in sera from patients at later stages of infection. These findings are open to several interpretations. On one hand, individual epitopes might be conformationally sensitive to reduction, for example, by the disruption of loop structures formed by disulfide bonds linking cysteine residues. On the other hand, it is possible that reduction so profoundly alters the overall molecular configuration of gpl20 that many epitopes are simply rendered inaccessible to antibody. The recent finding, noted above, that N70-1.9b binds to a synthetic peptide, which represents V3 and does not contain disulfide bonds linking cysteine residues, supports the latter possibility. A critical factor in HMAb production is the availability of an efficient and sensitive immunoassay for 575

ROBINSON ET AL.

screening hundreds of microwell cultures for antibody production. In one type of assay, which is the basis of most commercial HIV-1 ELISA kits used for sérologie screening, purified viral antigens are passively coated in wells of ELISA plates. This type of assay has also been used in some studies for HMAb screening. The preparation of antigens for this assay requires the production of very large amounts of virus which then must be purified and inactivated. The process of virus purification may result in significant losses of gpl20. Hence, this assay may be inefficient in detecting antibodies to gpl20 and favor detection of antibodies to other HIV antigens. This may explain in part the predominance of HMAbs reacting with gag proteins or gp41. The novel immunoassay in which HIV-1 glycoproteins released by infected cells grown in serum-free medium are affinity-immobilized in Con A-coated assay wells has greatly simplified the preparation of solid-phase glycoprotein antigens for large-scale antibody screening. The present results demonstrate the sensitivity and the high selectivity of this assay in detecting antibodies to gpl 20. Since virus does not have to be purified, only small volumes of cells grown in serum-free medium are needed to yield ample quantities of antigen for Con A immobilization. Indeed, many serum-free virus stocks can be diluted 1:2 or 1:4 without diminished antigen activity and thus, as little as 100 ml of supernatant fluid can be used to prepare 20-40 96-well ELISA plates. This method also simplifies the preparation of solid-phase glycoproteins from multiple strains of HIV as was demonstrated in the experiments shown in Figures 3 and 4. Theoretically, the binding of gp 120 to Con A could block access of antibodies to some epitopes. However, we found that murine monoclonals known to react either with the CD4 binding region or the V3 hypervariable domain (kindly provided by David Ho) react strongly with Con A-immobilized gpl20 (unpublished), indicating that epitopes within these two regions are represented in the assay. However, further testing with murine MAbs to other sites within gpl 20 is needed to determine if important epitopes are missing in this assay. We have not directly tested for gp41 binding to Con A. Another important aspect of monoclonal antibody production is the choice of virus strain used as the source of glycoprotein antigens for antibody screening. Since each HIV-1-infected B-cell donor may be immunized with epitopes of gpl20 that are not expressed by other strains of HIV-1, the ideal screening system should utilize antigens of the strain isolated from each B-cell donor. In the present experiments nonhomologous strains available in our laboratory at the time were used in screening. Nevertheless, since two of the four HMAbs produced react with variant epitopes, it is evident that antibody responses to some variant antigens can be probed using nonhomologous strains in the screening assay. On the other hand, we may have missed detecting antibodies binding to strain-restricted epitopes of the respective homologous strains. The preparation of solid-phase antigens from virus strains isolated from each asymptomatic B-cell donor presents a problem, since these strains generally do not replicate in continuous cell lines39 and usually can be propagated only in IL-2-dependent, activated primary T cells or in monocytes. The Con A glycoprotein immobilization assay offers a potential solution to this problem. As illustrated in Figure 3, one strain (L86) was grown in a serum-free culture of IL-2-dependent, activated primary T cells and gpl20 released into the medium functioned well in the Con A immobilization assay. If similar results can be achieved with other strains isolated from asymptomatic B-cell donors, it may become feasible to screen for antibodies reacting with antigens of homologous isolates. Our results raise the possibility that sizable numbers of HMAbs can be generated which cumulatively will provide a broad representation of human antibody responses to variant and conserved epitopes of gpl20 during asymptomatic infection. Such antibodies will be invaluable reagents in studies to define and map functionally important epitopes of gpl20 recognized by the human immune system. Moreover, neutralizing HMAbs to variant epitopes might be used to test sequential isolates from individual patients for the emergence of neutralization-resistant variant viruses. HMAbs may also reveal whether the same or separate epitopes are involved in neutralization and antibody-dependent virus enhancement. Information on this point would be particularly important in selecting epitopes that should be included in, or excluded from, candidate HIV-1 vaccines.

ACKNOWLEDGMENTS We thank Dr. Ronald Luftig for his support. This work was supported by a grant from the National Institutes of Health, NIH-NIAID-AI24030. The following reagents were obtained through the AIDS Research and

576

HUMAN MONOCLONAL ANTIBODIES TO HIV-1

gpl20

Reference Reagent Program, AIDS Program, NIAID, NIH: Sheep antiserum to HIV-1 gpl20 contributed by Dr. Michael Phelan, FDA; goat antiserum to HTLV-IIIB gpl60, contributed by AIDS Program, NIAID and produced under contract by Repligen;29 HTLV-IIIMN/H9, contributed by Robert C. Gallo;25-26 recömbinant gpl20 from HIV-1SF2, contributed Nancy Haigwood, Chiron Corporation.27

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early human

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