(~) INSTITUTPASTEUR/ELSEVIER Paris 1991

Res. Virol.

i99i, i42, 247-259

A human monoclonal antibody against the CD4-binding site of HIV1 gpl20 exhibits potent, broadly neutralizing activity S.A. Tilley (~) (*), W.J. Honnen

(l),

M.E. Racho

(l),

M. Hilgartner (2) and A. Pinter o)

(~J Public Health Research Institute, 455 First Avenue, N e w York, N Y 10016 (USA), and (2~ N e w York Hospital-Cornell Medical Center, 525 E. 68th St., New York, N Y 10021 (USA)

SUMMARY

A human monoclonai antibody (HuMAb) against H | V i , 1125H, was isolated from an asymptomatic, seropositive haemophiliac. This antibody was specific for ,3pl 20, and its binding to gp120 was inhibited by soluble CD4, indicating that its epitope was in or near the CD4-binding site. 1125H antibody recognized a variety of divergent HIV1 ~tra.~s, including most laboratory strains tested as well as some early passage isolates. Commensurate with its specificity and high apparent affinity, 1125H exhibited potent neutralizing activity against IIIB, MN, RF and SF-2 strains. The epitope recognized by 1125H was destroyed by reduction of disulphide bonds, but not by removal of N-linked sugars. Thus, the epitope was conformationally determined and did not involve carbohydrate. Data from radioimmunoprecipitation/SDS-PAGE analysis of proteolytically cleaved viral lysate further indicated that the epitope of 1125H was not affected by cleavage at the V3 loop of gp120, provided that gp120 disulphide bonds remained intact. The potential use of HuMAb 1125H in passive immunotherapy against HIV is discussed as well as the importance of including its epitope in an AIDS vaccine.

Key-words: HIV1, AIDS, Human monoclonal antibody, Virus neutralization, CD4, gp120; Binding site, Conformational epitope, Passive immunotherapy, Vaccine.

INTRODUCTION Earlier in the AIDS epidemic, the protective function of neutralizing antibodies against HIV was questioned, since such antibodies could be found in seropositive individuals who went on to develop AIDS. Now it is understood that the titres of neutralizing antibodies developed in humans during the course of HIV infection are generally not very high (Robert-Guroff et al., 1985; Weiss et al., 1985), that higher titres

Submitted April II, 1991, accepted May 23, 1991. (*) Correspondingauthor.

against certain epitopes do correlate with a better prognosis (Devash et al., 1990; Ho et aL, 1987; Ljunggren et al., 1987; Robert-Guroff et al., 1985 ; Rook et al., 1987), and that seropositive individuals may also possess deleterious antibodies against HIV that enhance viral infection (Homsy et al., 1988; Jouault et al., 1989; Robinson et al., 1990b; Takeda et al., 1988). Recent in vivo studies have demonstrated the protective effects of certain anti-HIV antibodies. In one such study, passive administration of

248

S.A. T I L L E Y E T A L .

hyperimmune plasma from healthy, HIVinfected humans to ARC and AIDS patients resulted in sustained clearance of p24 antigen and a maintenance or increase in the recipients' antiviral antibody titre, and clinical improvement was noted in 5 out of 9 recipients (Karpas et al., 1988). In another study, a correlation between the presence of high-affinity antibody against the principal neutralizing domain (V3 loop) of the MN strain of HIV1 and protection from vertical HIV transmission was observed (Devash et al., 1990). In animal studies, chimpanzees were challenged with IIIB strain of HIV1 that was previously incubated in vitro with neutralizing serum antibody from an HIV-seropositive cMr_qpanzee. The challenged animals were protected against ~5ral infection, as assessed by lack of viral replication and production of serum antibody to virus (Emini et al., 1990). More recently, Girard et al. have demonstrated successful longterm protection of 3 chimpanzees against HIV infection by immunization with recombinant gp ! 60 followed by a V3-1oop peptide (Girard et al., 1991). In another study, chimpanzees immunized with recombinant gp120 and challenged with HIV were protected from infection (Berm a n et al., 1990). In both of these vaccine trials, significant titres of strain-~necific neutrali7ino antibody were induced prior to challenge with virus. The protection obtained is believed to be due primarily to neutralizing antibody (Berman et al., 1990), since subunit vaccines are thought to be poor inducers of cytotoxic T cells. Prior to initiation of the studies described in thi~ report, it was accepted that humans produced broadly neutralizing antibodies against HIV (Matthews et al., 1986; Weiss et al., 1986), but the epitope specificity(ies) of these antibod-

BSA DTT EBV EH FITC HIV HuMAb

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bovine serum albumin. dithiothreitol. E p s t e i n - B a r r virus. endoglycosidase H. fluorescein i s o t h i o c y a n a t e . h u m a n i m m u n o d e f i c i e n c y virus. human monoclonal antibody.

ies was unknown. The principal neutralizing domain, or V3 loop, of HIV1 elicits predominately strain-specific antibodies in humans and experimental animals (Javaherian et al., 1989; Scott et aL, 1990). Attention was drawn away from the CD4-binding site of gpl20 as an epitope cluster eliciting broadly neutralizing antibodies because of studies showing daat human serum antibodies from HIV-infected individuals do not recognize linear peptides from this region (Sun et al., 1989), and it was believed that human antibodies against this region might not be of sufficient affinity to compete with the highaffinity gpl20/CD4 interaction (Lasky et al., 1987). In order to determine the epitope specificities and affinities of those human antibodies with broadly neutralizing activity against HIV, as well as those with other antiviral activities, we isolated and characterized human monoclonal antibodies (HuM A b) against the envelope glycoproteins of HIV1 from seropositive individuals. In this report, we describe a HuMAb against a conserved, conformational epitope in or near the CD4-binding site of HIV1 gpl20 that possesses potent neutralizing activity for a broad range of HIV1 isolates.

MATERIALS

AND

METHODS

Human subjects Peripheral blood was obtained for HuMAb isolation from HIVl-seropositive haemophiliacs classified as Walter Reed Stage 2A, i.e. having normal white blood cell counts and no history of opportunistic infections. These patients were followed at the Pediatric Hematology clinic at New York HospitalCornell Medical Center.

mAb Ig I-MMN NP-40 PBMC PBS

= = = = = =

monoclonal antibody. immunoglobulin. l-deoxymannojirimycin. N o n i d e t P-40. p e r i p h e r a l b l o o d m o n o n u c l e a r cell. p h o s p h a t e - b u f f e r e d saline.

B R O A D L Y N E U T R A L I Z I N G : H U M A N A N T ! B O D Y A G A I N S T ,I-1,,II,/1 g p l 2 0 HIV strains

HIV1 strains IIIB (Popovic et al., 1984; Ratner et al., 1985), SF2 (Levy et al., 1984; SanchezPescador et al., 1985), MN (Gallo et al., 1984; Gurgo et al., 1988) and RF (Popovic et ai., 1984; Starcich et al., 1986) have been described. The identities of strains IIIB, MN and RF were confirmed by us using strain-specific antisera raised against synthetic peptides corresponding to the V3 loop of each strain in an immunofluorescence assay. Two HIV 1 isolates, V-RF and PMS, were obtained from seropositive individuals in New York City by Dr. Fred Valentine, New York University School of Medicine, and were supplied in CEM cells. Two early passage HIV1 isolates, JR-CSF (Koyanagi et aL, 1987) and SM, were obtained from individuals in Los Angeles and San Francisco, respectively, and maintained in peripheral blood mononuclear cells (PBMC) by Dr. J.M. McCune, Systemix, Palo Alto, CA. An HIV2 strain, LAV2, has been described (Clavel et al., 1986).

Isolation of HuMAb against HIV env glycoproteins

PBMC were isolated by centrifugation of fresh, heparinized blood, diluted 1/3 with RPMI-1640 medium (Flow), on "Histopaque" (Sigma) at 400 g for 30 min at room temperature. Cells at the medium/Histopaque interface were recovered, diluted 7-8-fo!d with RPMI-1640 medium, and collected by centrifugation at approximately 400 g for 15 min. Cells were then washed by resuspension in 50 ml of RPMI-1640 medium, followed by centrifugation as above. The cell pellet was resuspended at a density of 2 x 106 cells/ml in RPMI-1640 medium supplemented with 15 070 (v/v) foetal calf serum (HyClone), 2 mM L-glutamine, penicillin (50 units/ml) and streptomycin (50 ~g/ml) (complete medium). Epstein-Burr virus (EBV) 100 x stock, prepared as described (Raubitschek, 1985) was then added so that it constituted 1/10 of the final volume of the cell suspension, and the cells were incubated overnight at 37°C in 5 °7o C O 2 in a 25 c m 2 flask. The following day. the cells were gently resuspended, diluted approximately 10-fold with RPMI-1640 medium, and collected by centrifugation. The pellet was resuspended at a final density of 10 4 cells/ml in complete medium. Cells were then plated in U-bottom 96-well plates at 100 l~l (1,000 cells) per well onto 100 ~tl of irradiated (3,500 rads) rat embryo fibroblasts in complete medium. Cultures were fed weekly for 4 weeks, at which time approximately 40 % of the wells exhibited growth. Their supernatants were then assayed for anti-env antibody production. Those cultures testing positive were picked onto fresh irradiated rat embryo fibroblasts in 96-well plates and re-assayed the fol-

249

lowing week. Cultures remaining positive were then sublined onto irradiated rat embryo fibroblasts at densities ranging from 1 to 100 cells/well. Those cultures growing in plates in which the number of wells with growth indicated > 95 07o probability of monoclonality as determined by the Poisson distribution (Coller and Coller, 1987) were re-tested for anti-env antibody production, and those testing positive were expanded into bulk culture. The monoclonality of these cultures was confirmed by Southern blot analysis. Southern blot analysis to determine clona!ity of cell lines

DNA isolation and restriction enzyme digestion, agarose gel electrophoresis, blotting to nitrocellulose, and hybridization to a 32p-labelled nick-translated probe were essentially as described (Eckhardt et al., 1982). DNA was cut with HindlII, which allows visualization of rearrangements due to V-D-J joining upon hybridization with an immunoglobulin JH region probe (Ravetch et ai., 1981). The JH probe used was a gift from Dr. Joel Buxbaum, New York University School of Medicine. It is an EcoRIHindlII fragment approximately 3.3 kb in !en~h from the germ line JH lOCUS; the HindIII site at its 3' end is present in the germline DNA (Ravetch et al., 1981), whereas the EcoRI site at its 5' end was created upon cloning (Buxbaum et aL, unpublished results). Clonal lines show only 2 J~hybridizing bands representing their two Ig heavy-chain loci. ELISA assays for HiV env-specific antibodies

The primary screening of EBV-transformed human cultures for production of anti-env antibody was done using recombinant gpl60 of BRU strain (construct no. 1163) (Kieny et a l , 1988) from Pasteur Merieux to coat PVC ELISA plates (Flow/ICN). In later assays on supernatants from cultures identified as positive in the initial screening, recombinant gpl20 (Genentech) (Leonard et aL, 1990); RP 135 (Rusche et al., 1988), a V3 peptide from the HIVBRU strain; V3 peptide (amino acids 305-328) from the HIVMN strain; gp41 (amino acids 560-641) (Centocor); gp41 (amino acids 647-665), or commercial HIV lysate (Organon-Technica or Dupont) was used to coat ELISA plates. Unless noted otherwise, 50 ng/well of protein diluted in Na2CO3/NaHCO3 buffer pH 9.8 was incubated in the plates overnight at 4°C. The following day, the plate was washed 3 times with PBS/Tween/azide (Sigma PBS with 0.05 °70 Tween-20, 1 mM NAN3), and the wells of the plate were blocked (to prevent non-specific binding) by incubation with 50 ~l of 2 070 BSA in PBS for !.5 h, 37°C. After washing, 50 ~tl of supernatant from hu-

250

S.A. T I L L E Y E T A L .

man cell lines or purified HuMAb or control sera diluted in 1 070 BSA/PBS was added to the wells and incubated as above. Following another wash, 50 izl of a 1/500 dilution of goat anti-human IgG conjugated to alkaline phosphatase (Zymed) in 2 070BSA was ~dded to each well. After an incubation and wash, 50 ~l of alkaline phosphatase substrate (disodium p-nitrophenyl phosphate), 1 mg/ml in diethanolamine buffer (1 M diethanolamine, 0.5 mM MgCI2, 3 mM NaN 3 pH 9.8) was added. The absorbance at 405 nm was read in a "Titertek Multiskan Plus" ELISA reader (Flow). The background obtained when culture media or diluent was used rather than human antibody was automatically subtracted from the results.

conjugated to alkaline phosphatase or goat antihuman lambda conjugated to alkaline phosphatase (Tago) (1/3,000) was used to detect bound HuMAb.

Measurement of apparent affinity constants The strength of binding of HuMAb to gpl60 was determined by diluting HuMAb of known concentration and assaying the various dilutions on gpl60-coated plates by ELISA as discussed above. It has been demonstrated that the concentration of antibody (expressed in nanomolar of half IgG molecules) at which half-maximal binding is observed is a rough value of 1/K (van Heyningen and van Heyningen, 1987).

Purification of HuMAb HuMAb were purified from cell supernatants on "MASS" protein A filters (Nygene Corp., Yonkers, NY) using "ImmunoPure" !gG binding and elution buffers (Pierce). The eluate (- 20 ml) was neutralized with 1/10 volume of 1 M Tris-HCl pH 8.0 and then dialysed against PBS. The antibodies were then concentrated using "CentriCell" ultrafilters (Polysciences, Inc.) to a final concentration of 0.3-1 mg/ml and stored at either 4°C, where they were stable over several weeks, or frozen in aliquots at - 1 3 5 ° C for lopg term storage. Quantitation of HuMAb •ncsc determinations were made by ELiSA using goat anti-human IgG (Zymed) (10 ~g/ml in 1 07o BSA in PBS) to coat the plates and capture the HuMAb in supernatants or purified antibody preparations (Gorny et al., 1989). The bound human antibody was detected with goat anti-human IgG conjugated to alkaline phosphatase (Zymed), and a standard curve was produced for each assay using affinity-purified human IgG (Cappel) of known concentration. Dc'.:=mination of HuMAb isotypes Heavy chain subclass was determined by an immunofluorescence assay in which HuMAb-producing cells were attached to slides and fixed with acetone. After blocking non-specific binding, the slides were reacted with human IgG-subclass-specific mouse monoclonal antibodies (mAb) (Zymed) (1/5000), followed by biotinylated goat anti-~ouse IgG (Zymed) (1/200), and then streptavidin-FITC (Zymed) (1/50). Light chain isotype was determined by an ELISA assay in which HuMAb were reacted with gpl60 attached to the plates, and then goat anti-human kappa

lmmunofluorescence assays for HIV strain specificity of HuMAb Cells that were maximally HIVl-infected or uninfected (either T-cell lines or PBMC) were washed in sterile PBS, resuspended in PBS at a density of 1-2 x 10 6 cells/ml, and incubated at 37°C for 30 min on "Multi-spot" microscope slides which had been pre-treated with )oly-L-lysine. The slides were then washed twice in PBS and once in dH20, fixed in acetone for 6-8 mins, and air dried. The slides with T-cell lines attached to them were reacted with supernatant from HuMAb-producing cell lines or dilutions of purified HuMAb or control serum in 1 mg/ml bovine gamma globulin in PBS for 1 h at 37°C~ After washing the d;a,~o..,,,,;,.o ;. vn~ ~,.a . . . . in dH20 and air drying, goat anti-human IgG conjugated to FITC (Zymed) (1/50) in 1 mg/ml bovine gamma globulin in PBS was incubated with the slides as in the previous step. The slides with PBMC attached to them were incubated with biotiay!ated HuMAb prepared essentially as described (Bayer and Wilchek, 1980) using antibodies purified as discussed above. IJzl~,~

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The binding of biotinylated antibodies was detected with streptavidin-FITC (Zymed, 1/50 dilution). After washing and air drying the slides as discussed above, the cells on the slides were counterstained with 0.05 07oEvans Blue for 10 min at room temperature. Following extensive washing with dH20 and air drying, 2 ~tl per well of 0.033 M DTT in 50 070 glycerol/PBS was added as preservative, a coverslip was added and the slides were viewed under a Nikon "Diaphot" immunofluorescence microscope.

Neutralization assay Purified HuMAb were diluted in complete media containing 10 °7o FCS to obtain various HuMAb con-

251

B R O A D L Y N E U T R A L I Z I N G H U M A N A N T I B O D Y A G A I N S T HIV1 gpl20

centrations in a total volume of 100 ptl° Included in this volume was 5 × t04 tO 2 X l0 s tissue culture infectious units of HIV 1, as assessed independently by end-point dilution of the virus. After a 30-min preincubation of virus and HuMAb at room temperature, the mixtures were each added to I x l0 s H9 cells in a final volume of 200 ld. Following a 24-h incubation at 37°C, the cells in each well were plated onto separate wells of poly-L-lysine-coated slides and stained sequentially with rat anti-nef serum (1/200) or rat anti-p24 serum (1/100) followed by rabbit antirat IgG conjugated to FITC (1/50) (Zymed). To detect RF-infected cells, staining was done with serum (1/200) from a seropositive individual followed by goat anti-human IgG conjugated to FITC (1/50) (Zymed). The used for staining antibodies were diluted in I mg/ml bovine gamma globulin in PBS. Cells were counterstained with Evan's Blue and the percentage of infected cells from each culture relative to the control (no mAb added) was assessed by counting immunfluorescent cells versus total counterstained cells under the fluorescence microscope. Using l0 s infectious units of virus per 105 H9 cells under the conditions of this assay, approximately 3 070of the cells (3,000 cells) were infected after 24 h in the absence of neutralizing antibody.

in PBS at 1/100 the original volume. After passage over a Sepharose CL-6B coiumn, the void volume containing virus was collected and the virus lysed at a final concentration of 0.5 °70 NP-40. Immunoblot strips were prepared essentially as described (PJ-~er et al., 1989), using loading buffer with or without 1 °7odithiothreitol (DTT). For deglycosylation experiments, virus-infected cells from which viral lysate was to be prepared were incubated either with or without 5 mM 1-deoxymannojirimycin (1-MMN) (Genzyme) to block the formation of complex type oligosaccharides from high mannose intermediates (Fuhrmann et al., 1984). The virus was purified on a Sepharose CL-6B column as described above, lysed in 1% SDS and boiled. A portion of this lysate was treated with an excess of endoglycosidase H (EH) (0.1 U/ml) (Boehringer-Mannheim) (Tarentino and Maley, 1974) for 2 h at 37°C. The samples were then diluted in loading buffer with 1 °70 SDS and no DTT prior to boiling and loading onto the gel. The Western blot strips were incubated with supernatants from humanantibody-producing cell lines or various dilutions of purified HuMAb or control sera, and bound antibody was detected with recombinant protein G conjugated to alkaline phosphatase (Zymed) (1/500).

RESULTS

RadioimmunaprecJpitation/SDS gel analyses Glycoproteins in HIVl-infected cells at 5-7 x l05 cell s/ml were labelled with 3H-glucosamine (10Q ~tCi/ml) as described (Pinter et al., 1989). Deper.ling on the experiment, either viral lysate or virusin[!cted cell lysate was prepared. For the latter, cells were lysed and immunoprecipitated as previously described (Pinter and Honnen, 1988). To prepare viral lysate, supernatant from virus-infected cells was adjusted to a final concentration of 0.5 °70NP-40 and 0.5 M NaCl using 20 x and 10 x stocks of the latter two reagents, respectively. The lysates were precleared with fixed, killed Staphylococcus aureus cells (Pansorbin) (Calbiochem), and then 70 ~l of virusinfected cell lysate or 140 ~tl of viral lysate was added to an equal volume of supernatant from human antibody-producing cell lines, or various dilutions of purified HuMAb or of control sera. Following incubation ~nd precipitation by Pansorbin, the pellet was brought up iii ~m-~.p!abuffer containing I 070DTT and run on_ an ! ! % po!yacrylamide gel as described (Laemmli, 1970). Fluorography (Bonner and Laskey, 1974) then allowed detection of radiolabelled, immunoprecipitated glycoproteins in the gel.

From a healthy, seropositive haemophiliac, we isolated an EBV-transformed, monoclonal line producing H u M A b 1125H (T1, x) that was

Immunoblotting analyses

The i 125H antibody recognized a variety of divergent HIV1 strains as assessed by specific binding to virus-infected cells in an im-

To prepare viral lysate, virus was pelleted ~rom the HIV-infected cell supernatant and resuspended

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bee.~ in continuous culture for over 16 months and is stable in terms of H u M A b production. Supernatants of dense cultures ( 0 . 5 - 1 x 10 6 cells/ml) of 1125H cells typically contain approximately 2 l~g/ml of HuMAb, which is characteristic of EBV-transformed cell lines (Crawford, 1985). The specificity of H u M A b 1125H was evidenced by its reactivity with recombinant gp 160 and recombinant gpl20, but not with a variety of other peptides, including those from gp4!, in ELISA (Tilley et al., 1991). This specificity is further documented in figure 1, which shows that 1125H specifically immunoprecipitated gpl20 and gpl60, but not gp41, from lysates of H9 cells infected with the IIIB and RF isolates of HIVI.

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Fig. 1. R~dioimmunoprecipitation/SDS gel analysis of lysates from 3H-glucosamine-labelled HIVl-infected H9 cells. Lysate from either IIIB- or RF-infected cells was precipitated with a human serum 0/400 dilution) from an HIVl-seropositive individual (lane l) or l0 Fg/ml purified 1125H HuMAb (lane 2). The precursor, surface and transmembrane viral glycoproteins (Prenv, SUenv, TMe,v) from the IIIB strain are approximately 160, 120 and 41 ":D, respectively, while the corresponding glycoproteins of the RF strain are of lower molecular weight, approximately 147, 113 and 34 kD, apparently due to glycosylation differences between the strains. Molecular weight markers were prestained standards ranging from 14.3 to 200 kD (BRL).

munofluorescence assay (data not shown). The characterized laboratory strains IIIB, RF, MN and SF-2 were all recognized by 1125H. Also, one (V-RF) of two New York isolates of HIV1 adapted to growth in a T-cell line, CEM, as well as one of two early passage isolates grown only in PBMC, i.e. JR-CSF (Koyanagi et aL, 1987), were reactive with 1125H. 1125H HuMAb did not react with LAV2, an HIV2 isolate. Thus, 1125H recognizes a proportion of early passage isolates of HIV1 as well as HIV1 strains repeatedly passaged in T-cell lines. The broad strain specificity of 1125H antibody suggested that the antibody might recognize an epitope in or near the CD4-binding site of gpl20, since this region would be expected to be relatively conserved across HIVI strains (Lasky et al., 1987). To address this question, we asked whether soluble, recombinant CD4

Figure 2 shows that CD4 inhibits the binding of 1125H to gpl60 in a concentrationdependent manner. Under the conditions of this experiment, approximately a 7-fold molar excess of CD4 to 1125H was required to achieve 50 07o inhibition of 1125H binding. Similar results were obtained when recombinant gpl20 was used in the assay rather than recombinant gpl60 (data not shown). The binding of our anti-gp41 HuMAb, 31710B (T1,),), to gpl60 was not inhibited by CD4 (fig. 2), demonstrating that the CD4 preparation did not contain non-specific inhibitory activity. The apparent affinity of 1125H for its epitope was measured by solid-phase ELISA using gpl60 attached to plates, and was found to be approximately 1.3 x 10 9 l/mole (data not shown). This affinity is higher than that estimated for the binding of HIV1 gpl20 to CD4, 2.5 x 108 l/mole (Lasky et al., 1987), indicating that 1125H should effectively compete with the CD4 receptor on cells for binding to virus. This concept was tested by assessing the ability of 1125H to neutralize infectivity of various strains of HIV1 in vitro in a 24-h flncJre~eent f n e n ¢ assay. Figure 3 shows that 1125H exhibits potent neutralizing activity against 4 laboratory strains of HIVI tested, including the MN isolate of HIV1 which is highly similar to most primary isolates from the United States based on V3 sequences (LaRosa et al., 1990). The results show 50 °70 neutralization of 10 5 infectious units of IIIB and MN strains at 0.5-1 l~g/ml of 1125H, while approximately 5 ~g/ml of 1125H was required to achieve the same level of neutralization of RF and SF-2 strains. For comparison, affinity purified anti-V3BRU antibody from serum of a hyperimmunized chimpanzee (no. 433) (Girard et al., 1991) exhibited 50 070 neutralization of 105 infectious units of the IIIB strain at approximately ,,.,. n ,) ~ . . , / ,,.,,, ~ ... ;, our neutrahzatlorl " " assay (data not shown). This neutralizing antibody appears to have been largely responsible for the partial protection against HIV challenge

B_OC)ADI Y N E U T R A /_./ZING H U M A N A N T I B O D Y A G A I N S T HIV1 gpl20

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[CD4], jug/ml Fig. 2. Results of an ELISA assay assessing the effect of soluble CD4 on binding of HuMAb to gpl60. Purified HuMAb were combined with different concentrations of recombinant CD4 in 1 % BSA/PBS at a constant volume. The final concentration of HuMAb was 0,07 izg/ml, while the final concentrations of CD4 are shown in the figure. HuMAb/CD4 mixtures were plated onto gpl60-coated ELISA plates (50 Izl per well), and bound H u M A b was detected with goat anti-human IgG coupled to alkaline phosphatase. After alkaline phosphatase substrate addition, the reactions were allowed to continue until the absorbance at 405 nm in the control wells (with no CD4 added) reached an OD of 0.7. The error bars shown for 1125H HuMAb represent the standard deviation of triplicate

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thin w~ observed in chimpanzee no. 433 (Girard et al., 1991). Our anti-gp41 HuMAb, 31710B, exhibited no neutralizing activity (fig. 3) and served as a negative control in the assay.

Further experiments were done to characterize the epitope recognized by 1125H. Immunoblot analysis of reduced versus non-reduced viral lysates showed that 1125H loses reactivity with

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Yig. 4. Immunoblotting analyses of lysate from purified HIV1 after various treatments. Panel A shows lysate prepared from the MN viral strain and Loiled in loading buffer without DTT (lanes I and 2) or with 1 % DTT (lanes 3 and 4). Immuno[~iot strips prepared in this way were reacted with either rat antiserum against gpl20 (1/250 dilution) (lanes 1 and 3) er 20 l~g/ml 1125H HuMAb (lanes 2 and 4). Panel B shows lysate from the IIIB viral strain prepr.red from virus-infected cells that had been grown either in the presence or ab~:ence of I-MMN. A proportion of the lysates was then treated with EH, while another proportion was not. Immunoblot strips prepared from lysates so treated were reacted with rat antiserum against gp 120 (1/250 dilutioh) tiane 1), cell supernatant containing 1125H HuMAb (1/2 dflutiGn) (lane 2), or purified 1125H HuMAb (approximately 20 i~g/ml) (lane 3). The molecular weight markers ~ece prestained standards ranging from 14.3 to 200 kD (BRL).

gpl20 and gpl60 upon disruption of their disulphide bonds (fig. 4A), indicating that the epitope of 1125H is conformationally determined. In order to assess whether the epitope was dependent on N-linked glycans, we removed the N-linked sugars from gpl20 under conditions that would not disrupt disulphide bonds. To achieve this, we isolated virus from cells grown in the presence of 1-MMN, a srbstance that inhibits the processing of high mannose sugars to complex carbohydrates (Fuhrmann et al., 1984). The high mannose sugars, unlike complex carbohydrates, are sensitive to digestion with EH, which acts efficiently on molecules that have been denatured in the absence of reducing agents. Complete digestion of these N-glycans with EH results in a product with a single N-linked sugar residue (N-acetylglucosamine) remaining at each g!ycan attachment site on the protein (Tarentino and Maley, 1974). Figure 4B

shows that HuMAb 1125H recognized gpl20 and gp 160 produced in the presence of I-MMN both before and after removal of the glycans with EH, indicating that carbohydrate is not an essential component of the 1125H epitope. Finally, we observed reactivity of 1125H with gpl20 that had been proteolytically cleaved at the V3 loop. Figure 5 shows that 1125H immunoprecipitates not only intact gpl20 and gpl60 from radiolabelled viral lysates, but also proteolytic fragments of 47 and 73 kD, which are of approximately the same size as those previously shown (Clements et al., 1991) to be generated by cleavage at the V3 loop of gp 120 either spontaneously or by treatment with various proteases. This result indicates that cleavage of the V3 loop does not interfere with the integrity of the 1125H epitope. Since 1125H recognizes an epitope in or near the CD4-binding site, our result correlates with the observation that

BROADLY NEUTRALIZING AO

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Furthermore, our results indicate that spontaneous cleavage of the V3 loop of viral gpl60 also occurs, since fragments of the size predicted to be generated by such a cleavage, i.e. 73 and 87 kD, were recognized by anti-gp41 HuMAb 31710B, in addition to intact gpi60 ann gp4i molecules (lane 4). It has been suggested that cleavage of the V3 loop by cell surface or endosomal proteinases is important for HIV-celi fusion (Clements et al., 1991).

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255

l

41 31

Proteolytic cleavage

eo

DISCUSSION I/~ loop

I,

I

'a~

gpl60 I

s:ka

I

"-'i

I

Fig. 5. Radioimmunoprecipitation/SDS gel analysis of lysed HIVI from supernatants of 3H-glucosamine-labelled cells infected with the IIIB strain of virus. tated with serum from a seropositive individual (1/500 dilution) (lane 1), affinity-purified anti-V3nRu serum antibodies from a chimpanzee (no. 433) immunized with gpl60 and V3BRU peptide (Girard et al., 1991) (20 ~g/ml) (lane 2), cell supernatant containing 1125H HuMAb (1/2 dilution) (lane 3), or cell supernatant containing 31710B HuMAb (1/2 dilution) (lane 4). The molecular weight markers were 35S-cysteine-labelled HIVI proteins immunoprecipitated with serum from a seropositive individual (results not shown). Panel B is a diagram mapping the gpl20- and gpl60-derived proteolytic fragments of the apparent molecular weights immunoprecipitated by envspecific antibodies shown in panel A.

V3-1oop cleavage does not affect binding of recombinant gpl20 to CD4 (Clements et al., 1991). The fact that cleavage occurred at the V3 loop in the experiment shown here (fig. 5) was confirmed by the lack of reactivity of affinitypurified chimpanzee anti-V3 loop antibodies with either of the proteolytic fragments (lane 2), though they reacted with intact gp 120 and gp 160.

Our isolation of a neutralizing HuMAb against a conformational epitope in or near the CD4-binding site of gpl20 from a heathy, HIVinfected individual is significant in several respects. First, our data, in conjunction with that of two other groups (Hoet al., 1991 ; Posner et al., 1991), demonstrate for the first time that humans can produce broadly neutralizing antibodies against the CD4-binding site. Robinson, Ho and coworkers have recently isolated and characterized a HuMAb, 15e, against the CD4-binding site and have demonstrated that serum antibodies competing with 15e are detectable in humans several months after seroconversion (Hoet al., 1991 ; Robinson et aL, 1990a). The epitope~ of 1125H and 15e are distinct, however, as evidenced by 1125H reactivity with and neutralization of the RF Haitian isolate of HIVI not recognized by 15e ( H o e t al., 1991). Also, Posner et al. recently isolated a HuMAb, F105, against a conformational epitope of the CD4 binding site (Posner et al., 1991). The epitopes of FI05 and our HuMAb 1125H are distinct based on the fact that i 125H exFdbits potent and similar neutralization of the MN and IIIB strains of HIVI, whereas F105 exhibits significantly greater neutralization of IIIB than MN. Thus, the CD4-binding site, and perhaps adjacent regions of gpl20, present a cluster of conformational epitopes to which humans can respond with high affinity, broadly neutralizing antibodies. The epitope of 1125H, though conformationally determined, appears to be independent of carbohydrate. It is not entirely clear whether

256

S.A. T I L L E Y E T AL.

N-linked carbohydrates on gp 120 are important in CD4 binding, although some contribution of carbohydrate to this interaction has been suggested (Fenouillet et al., 1990; Matthews et al., 1987). Thus, the CD4-binding site of gpl20 may not be identical to the 1125H epitope. Indeed, preliminary evidence from reactivity of 1125H with a series of gpl20 mutants indicates that the 1125H epitope overlaps that of the CD4-binding site but is not identical to it (Sodroski et al., unpublished results; Olshevsky et al., 1990). In order to present the 1125H epitope in its most immunogenic form in a vaccine, it may be necessary to separate it physically a n d / o r temporally from the immunodominant V3 loop. This conclusion can be drawn from studies where immunization of chimpanzees with recombinant gpl60 or gpl20 elicited predominately typespecific antibody against the V3 loop and little, if any, broadly neutralizing antibody against the CD4-binding site (Berman et al., 1990; Girard et al., 1991). The lack of response to the CD4-binding site epitc, pes was apparently not due to paucity of these epitopes in the recombinant proteins, since these proteins bind to CD4, and we demonstrated 1125H reactivity with these recombinant protaln~

Thp

r a a t - t i v i t ~ a r~f 1 1 9 ¢ I . - I u , ; * h , , ~ l ' ) t ~ , h , ~ ,

has been proteolytically cleaved at the V3 loop may indicate a starting point for creating an immunogen depleted in V3 epitopes that presents immunogenic CD4-binding-site epitopes. Such a subunit vaccine could be given in conjunction with other constructs that independently present the V3 loop. Finally, while the in vivo neutralizing activity of the 1125H H u M A b has yet to be assessed, the potent, broadly neutralizing activity of this H u M A b in vitro suggests the possible utility of this antibody as a prophylactic or therapeutic agent against HIV. Since it is of human origin, this antibody should possess low immunogenicity in humans. Administration of such neutralizing antibodies may be particularly useful in preventing infection after acute exposure to HIV and in blocking maternal/foetal HIV transmission (Devash et al., 1990). Under appropriate conditions, these reagents may find applications in

the treatment of chronically infected individuals as well.

Acknowledgements We thank R. Neurath for rabbit anti-V3 antisera; M. Girard and E. Muchmore for the chimpanzee anti-V3 antiserum; F. Valentine and J.M. McCune for HIVI isolates; M. Girard and M. Kaczorek for recombinant gp 160 and V3BRu peptide; D. Konys for recombinant CD4; J. Buxbaum for the JR probe; D. Cook for assistance in immunofluorescence assays; and M. Gorny and S. ZollaPazner for helpful discussions. This work was supported by NIH grants AI26081 (to S.A.T.) and AI23884 and AI27742 (to A.P.).

Activit~ neutralisante puissante et Etendue d'un anticorps monoclonal humain anti-site liant le CD4, d'Ag~ contre la gp120 du VIH1

Un anticorps monoclonal humain 1125H (mAbHu) anti-VIH 1 a 6t6 isol6 d'un h6mophile s~ropositif asymptomatique. Cet anticorps est sp~cifique de la gpl20 et sa liaison/l la gpl20 est inhib~e par la molecule CD4, ce qui indique que son ~pitope se trouve dans ou pros du site liant le CD4. Cet anticorps mAbHu 1125H reconnaTt des souches VIH1 diff6rentes, y compris la plupart des souches de laboratoire et des souches isol~es en passage pr~coce. Proportionneiiement/~ sa sp~cificit~ et ~t sa forte affinit~ apparente, l'anticorps 1125H montre une activit6 neutralisante puissante/l l'6gard des souches IIIB, MN, RF et SF-2. L'6pitope reconnu par le mAbHu 1125H est d&ruit par r6duction des ponts disulfure, mais non par suppression des sucres N-li6s. Ainsi l'6pitope est d6termin6 sur le plan conformationnel et ne semble pas impliquer d'hydrate de carbone. Les r6sultats de l'analyse, par radioimmunopr6cipitation/61ectrophor~se en gel de polyacrilamide en pr6sence de sodium-dod6cyl-sulphate, du lysat viral obtenu par clivage prot6olytique, indiquent de plus que l'6pitope du mAbHu 1125H n'est pas affect6 par clivage/l la boucle V3 de la gpl20 pourvu que les ponts disulfites de la gpl20 restent intacts. L'utilisation potentielle du mAbHu 1125H en immunoth& rapie passive contre le VIH1 est discut6 ainsi que l'opportunit6 d'inclure cet 6pitope dans le vaccin contre le SIDA. Mots-cl6s: VIHI, SIDA, Anticorps monoclonal humain, Neutralisation virale, CD4, gpl20; Site de liaison, Epitope conformationnel, Immunoth6rapie passive, Vaccin.

BROADL Y NEUTRALIZING

HUMAN

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