Vol. 64, No. 6
JOURNAL OF VIROLOGY, June 1990, p. 2833-2840
0022-538X/90/062833-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Inhibition of Human Immunodeficiency Virus (HIV) Infection In Vitro by Anticarbohydrate Monoclonal Antibodies: Peripheral Glycosylation of HIV Envelope Glycoprotein gpl20 May Be a Target for Virus Neutralization JOHN-ERIK S. HANSEN,1* HENRIK CLAUSEN,2 CLAUS NIELSEN,3 LARS S. TEGLBJAERG,1 LISBETH L. HANSEN,4 CARSTEN M. NIELSEN,3 ERIK DABELSTEEN,S LARS MATHIESEN,1 SEN-ITIROH HAKOMORI,2 AND JENS 0. NIELSEN1 Department of Infectious Diseases 144, Hvidovre Hospital, 2650 Hvidovre, Denmark1; The Biomembrane Institute,
Seattle, Washington2; and Enterovirus Department, State Serum Institute,3 The Danish Cancer Society, Fibiger Institute,4 and the Royal Dental College,5 Copenhagen, Denmark Received 18 October 1989/Accepted 14 March 1990
Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N- and 0-linked carbohydrate epitopes (Ley, Al, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLV111B or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 1251-labeled gpl20. The MAbs therefore define carbohydrate structures expressed by the viral envelope glycoprotein gpl20, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both. a MAb (BM1) directed to the Ley epitope, after HIV infection (3). The possibility thus exists that novel glycan structures may be produced by HIVinfected cells and incorporated into the viral envelope. We have utilized a panel of MAbs with well-defined anticarbohydrate specificity for inhibition of HIV infectivity to determine whether glycan epitopes could be used as targets for virus neutralization.
ture, recognized by
Altered glycosylation in host cells associated with viral infection has been reported (24, 41). Like oncogenesis, aberrant glycosylation induced by cytomegalovirus or by human immunodeficiency virus (HIV) causes formation of neoantigens which are absent in the original host cells. By using monoclonal antibodies (MAbs) which define oligosaccharide epitopes, the appearance of Lex and Ley antigens after viral infection has thus been detected (3, 5). Several studies have indicated the involvement of the carbohydrate part of HIV in in vitro infection. Thus, inhibition of the early steps in Golgi glycosylation in infected cells reduces the infectivity of the virus produced (16, 31), and lectins block syncytium formation, probably by a specific interaction with gpl20 glycans of infected cells, and also neutralize infectivity of cell-free virus (18, 27). Variation in N-glycosylation of target T4 cells, however, does not seem to influence susceptibility to HIV infection (31). gpl20 contains several different glycan structures, and carbohydrate constitutes 50% of the total mass of gpl20 (29). So far, only N-linked glycans have been found on gpl20 (23). The binding site on gpl20 for the T4 receptor seems to be located in a nonlinear C-terminal part of the molecule (26). Whether glycan(s) participates directly in virus binding is not clear. Thus, inhibitory lectins may bind to glycans adjacent to the binding site and thereby sterically interfere with T4 gpl20 binding (18), as has been found for neutralizing antibodies (6, 28). Glycans so far identified on gpl20 by lectin studies are rather ubiquitous, and the therapeutic potential of lectin-based treatment therefore seems small. In a study of neoantigen expression on virus-infected cells, T4 cells were found to express a novel glycan struc*
MATERIALS AND METHODS Anticarbohydrate MAbs. The specificities, isotypes, and references for production of MAbs used in this study are listed in Table 1. All hybridoma cultures were grown in RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, and 1 mM pyruvate, and were stored at 4°C with 0.02% NaN3. The study was performed in three stages. Initially, all listed MAbs were tested for HIV inhibition as culture hybridoma supernatants containing 10 to 50 jig of immunoglobulin per ml. In the second stage, MAbs showing some reduction of HIV infection (TKH2, BM1, AH21, AH16, NKH1, HH6, SH2, and CA3F4) were retested after extensive dialysis of the hybridoma supernatants against phosphate-buffered saline (PBS) to remove preservatives. Finally, the MAbs TKH2, B72.3, BM1, and AH21 were used after purification from culture supernatants. TKH2 and B72.3 were purified by protein A-Sepharose chromatography, and AH21 and BM1 were purified by ion-exchange chromatography. Cells. For HIV infection experiments, the T4 lymphocyte cell lines H9 (40), CEM (13), and MT4 (19) were cultured at 37°C in 5% CO2 by using RPMI 1640 with 10% fetal calf serum (5% for CEM cells), 2 ,uM glutamine, 100 IU of penicillin per ml, 20 ,ug of gentamicin per ml, and 100 IU of
Corresponding author. 2833
TABLE 1. Antigen specificities and references for MAbs used in this study
Type 1 Lea
Gall1-3GIcNAcpl--*R 4 T
AH21 (IgM) AH16 (IgG3) HH6 (IgG2a)
338 338 (Clausen, unpublished data
SH1, -2 (IgG)
sSinghal, unpublished data
(E. D. Nudelman, U. Mandel, S. B. Levery, T. Kaizu, and S. Hakomori, J. Biol. Chem., in press)
Nudelman and Mandel, unpublished data
Galpl-*3GlcNAcpl-*R 2 /Fc Fucotl
Type 2 N-Lac H
Lex (ext. Lex)
AH16, HH6: see A, 3
Galp1-4GlcNAcpl-->R 2 A
Type 3 Tn Sialyl Tn
Lu35 (IgM) TKH2 (IgGl) B72.3 (IgGl)
20 22 35
HH8 (IgM) HH14 (IgM)
11 Clausen, unpublished data
TKH7 (IgM) TKH5 (IgM)
Kjeldsen, unpublished data Kjeldsen, unpublished data
Type 4 T,B
Galpl1-3GaINAcpl-4GalplB13R Gal1l-3GalNAc l14Galpl1-4GIclc,SCer 2 6 T T
VOL. 64, 1990
HIV INHIBITION BY CARBOHYDRATE-SPECIFIC MAbs
streptomycin per ml (growth medium). Cells were maintained at a concentration of 2 x 105 to 10 x 105 cells per ml, and medium was exchanged twice weekly. Peripheral blood mononuclear cells (PMC) were obtained from healthy donors by Ficoll-Hypaque gradient centrifugation (7), and T4 lymphocytes were selected by panning (46). Donor PMC and T4 lymphocytes were cultured as described above in growth medium supplemented with 20 IU of interleukin-2 (Boehringer GmbH, Mannheim, Federal Republic of Germany) per ml. Before infection with HIV, donor lymphocytes (PMC or selected T4 cells) were stimulated for 3 days with 5 ,ug of phytohemagglutinin (PHA-P; GIBCO, Uxbridge, England) per ml. Virus. A supernatant from H9 cells chronically infected with the reference HIV-1 strain HTLVIIIB (40) was sterile filtered, aliquotted, and stored at -80°C until use. The HIV-1 strain SSI-002, isolated from an HIV-infected patient (Centers for Disease Control, stage II) as previously described (C. Nielsen, J. Larsen Petersen, C. M. Nielsen, C. Pedersen, L. R. Mathlesen, and B. F. Vestergaard, J. Virol. Methods, in press), was passed three times in MT4 cells and stored as described above. Before use, the 50% tissue culture infective doses (TCID50s) of the virus preparations were determined in MT4 cells and PMC. Virus inhibition assay. Ten TCID50s of HTLVIIIB were mixed with 0.5 ml of hybridoma supernatant and incubated for 1 h at 37°C. MT4 cells (1 x 106) were suspended in this mixture and incubated for 2 h at 37°C. After extensive washing, the cells were suspended in 4 ml of growth medium containing 10% (vol/vol) of the corresponding hybridoma supernatant and duplicate 1.5-ml cell suspensions were transferred to a 24-well cell culture plate. The cells were cultured for 7 days at 37°C in 5% C02, and 750 RI of cell-free supernatant was exchanged with fresh medium without hybridoma supernatant after 2, 4, and 7 days. HIV antigen output was measured by enzyme-linked immunosorbent assay (ELISA) of the supernatants. Dialyzed hybridoma supernatants (200 pul) were preincubated with 10 TCID50s of HTLVIIIB or SSI-002 before inoculation and culture as described above. Purified MAb was preincubated with 10 TCID50s of B, which was then used to inoculate MT4 cells (1 x HTLV,,, 106), PMC (4 x 106), or selected T4 lymphocytes (4 x 106). After incubation for 2 h at 37°C, the cells were washed and resuspended in 5 ml of growth medium. Quadruplicate 1-ml cell suspensions were transferred to a 24-well cell culture plate. The supernatant (500 ,ul) was exchanged with fresh growth medium after 4 and 7 days of culture. Syncytium assay. Ten thousand permissively HTLVIIIBinfected H9 cells were incubated with 7.5 pug of MAb in 50 pul of growth medium for 1 h in wells of a 96-well cell culture plate. Then 105 CEM cells in 50 pu1 of growth medium was added. After incubation for 24 h, all syncytia (giant cells with more than 5 nuclei) in each well were counted. Live cell counts were obtained by trypan blue exclusion. ELISA. Cell-free culture supernatants were examined for HIV antigen by using a double-antibody sandwich ELISA as described previously (33). Each plate included a dilution series of a standard HIV antigen preparation, and optical densities (490 nm) were expressed relative to this standard preparation (arbitrary units). All in vitro infection experiments included a control in which untreated HTLVIIIB was used. After 4 days of culture, supernatants were diluted to give an optical density at 490 nm from this control of approximately 2. All antibodies tested for inhibition of
infection were also tested for interference with the ELISA detection, and no such interference was found. Toxicity. Toxicity of the MAb preparations was examined by incubating 0.5 x 106 MT4 cells in 24-well cell culture plates in growth medium containing 20 ,ug of purified antibody per ml (maximal concentration used in infectivity experiments). Live-cell counts were obtained on days 0 and 4 by using trypan blue exclusion. Polymerase chain reaction and dot blot hybridization. Cell pellets were suspended in 100 pl of 1% Triton X-100 in PBS and boiled for 10 min to release DNA. A 10-,ul portion was mixed with 90 ,ul of amplification mixture (200 pmol of each primer; 200 pumol each of dATP, dCTP, dGTP, and dTTP; 50 mM KCl; 10 mM Tris [pH 8.3]; 6.5 mM MgCl2; and 0.02% gelatin). The primers used were SK29/SK30 (37), kindly supplied by H. D. Andersen, Novo Nordisk, Denmark. The mixture was heated to 94°C for 5 min, and then 2 U of Taq polymerase (The Perkin-Elmer Corp., Norwalk, Conn.) was added. Forty rounds of amplification were carried out in a DNA thermal cycler (Perkin-Elmer). Each cycle lasted 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. A 1-pul portion of the amplified product was mixed with 60 plA of 0.67 M NaOH-0.67 M NaCl and boiled for 5 min, and this procedure was followed by cooling on ice. Samples were transferred to a Zeta-probe membrane in a vacuum manifold, and sample wells were rinsed with 500 pA of 0.4 M NaOH each. The membrane was rinsed briefly in 2 x standard saline citrate solution (SSC; lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0), air dried, and heated at 80°C for 2 h. Membranes were prehybridized for 2 h at 42°C in hybridization solution (50% deionized formamide, 1% sodium dodecyl sulfate [SDS], 1 M NaCl, 50 mg of dextran sulfate per ml, 0.1 mg of sheared salmon sperm DNA [Sigma Chemical Co., St. Louis, Mo.] per ml). Subsequently, 20 pmol of 32P-labeled probe (SK31) was added, and hybridization was performed overnight at 42°C. Membranes were washed for 10 min in 2 x SSC-0.1% SDS at room temperature, and this procedure was followed by 30 min in 2x SSC-0.1% SDS at 62°C. Finally, the membrane was rinsed two times for 15 min each time in 0.2x SSC-0.1% SDS at room temperature and exposed to Kodak XAR films overnight. Immunofluorescence analysis. Binding of the MAbs to cells was determined by fluorescence-activated cell sorting (FACS). Briefly, 106 cells suspended in 50 pA of suspension buffer (PBS containing 1% bovine serum albumin, 2% human serum, and 0. 1% azide) were incubated with 50 pA of hybridoma supernatant for 45 min at 4°C. The cells were then washed once in 1 ml of cold PBS and incubated for another 30 min at 4°C with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Copenhagen, Denmark) diluted 1:30 in suspension buffer. After incubation, the cells were washed once and were suspended in 1 ml of PBS with 1% paraformaldehyde. The cells were analyzed by flow cytometry by using a fluorescence-activated cell sorter (FACS-SCAN; Becton Dickinson Labware, Oxnard, Calif.). MAbs against CD4 (Leu3a; Becton Dickinson) and against gpl20 (Du Pont, Stevenage, England) were used as controls. Radioimmunoprecipitation assay. A sucrose gradient-purified HTLVIIIB virus stock, kindly provided by Robert C. Gallo (National Institutes of Health, Bethesda, Md.), was lysed in 0.5% Triton X-100-0.5 M KCI to a final protein concentration of 0.12 mg/ml. A 20-R.g portion of this lysate in a total volume of 260 RI1 was iodinated by using the chloramine T method. The protein was mixed with 1 mCi of 1251 (IMS.30; Amersham, Little Chalfont, England) and 40 jig of
HANSEN ET AL.
chloramine T (N-chloro-p-toluenesulfonamide sodium salt, 2 mg of stock solution per ml in distilled water, freshly prepared) in a 50 mM Tris hydrochloride buffer, pH 8.0-1% Nonidet P-40. After 2 min, the reaction was stopped by adding 10 ,u of sodium disulfite (10 mg/ml in distilled water) and 50 ,ul of 0.1 M KI. The iodinated protein was separated from unreacted iodide by gel filtration on a prepacked PD-10 column (Pharmacia, Uppsala, Sweden). The column was eluted with TEN buffer (20 mM Tris hydrochloride [pH 8.0], 1 mM EDTA, 100 mM NaCl) with 1% Nonidet P-40. The fraction containing the protein was clarified by centrifuging for 30 min at 20,000 x g. The protein was aliquotted and stored at -80°C. One ,uCi of 1251-labeled HIV lysate was incubated with MAbs or serum in lysis buffer (100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 100 IU of aprotinin per ml, 20 mM Tris hydrochloride [pH 8.0]) for 3 h at 4°C in a total volume of 200 ,ul. When the catching antibody was of immunoglobulin class G (IgG) (AH16, TKH2, and control MAb SH1), 100 ,ul of 10% protein A-Sepharose (Pharmacia) in lysis buffer was added and the mixture was incubated overnight at 4°C on a rotor. When the catching antibody was IgM (BM1 and control MAb FH7), the protein A-Sepharose was preincubated overnight with goat anti-mouse IgM (Sigma) and washed with lysis buffer containing 1 mg of bovine serum albumin per ml. The pellet was washed once in DOC buffer (500 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 30% sucrose, 1% deoxycholate, 20 mM Tris hydrochloride [pH 7.6]) and twice in 10 mM Tris-NaCl buffer, pH 7.6. The pellet was suspended in 50 ,u1 of sample buffer (2% [wt/vol] SDS, 0.05 g of dithiotreitol, 0.2% [wt/vol] tetramethylethylendiamin, 0.01% [wt/vol] Pyronine Y, and 10% [vol/vol] glycerol in 0.4 M Tris-H2SO4 buffer [pH 7.2]) and incubated at 100°C for 3 min, and the supernatant was used for SDS-polyacrylamide gel electrophoresis as previously described (18) in 8 or 10% homogeneous gels. Molecular weight standards were 14C-labeled proteins from Amersham. Gels were dried and put on Kodak XAR films for 7 to 30 days.
Arbitrary units Day 2
NoAb NoHIV NKH1 HH6 TKH2 SH2
AH21 AH16 CA3F4
Hybridoma supernatants FIG. 1. HIV antigen in culture supernatants. HTLVIIB was preincubated with dialyzed hybridoma supernatants containing antibodies against defined carbohydrate antigens and used to infect the T-cell line MT4. At 2, 4, and 7 days, HIV antigen was measured in the culture supernatants. Means of duplicate experiments are shown. NoAb, Control with untreated virus; NoHIV, control without virus. Cells from HIV antigen-negative cultures were also negative for HIV DNA by polymerase chain reaction, and cells from HIV antigen-positive cultures were also positive for HIV DNA by polymerase chain reaction.
Percent inhibition with MAb AH21
Percent Inhibition with MAb BM1
60 60-40 _ 40-
RESULTS Twenty MAbs against various carbohydrate epitopes (Table 1) were tested for their ability to inhibit HIV infection of MT4 cells in vitro. The MAbs defined carbohydrate structures located at the periphery of both N-linked and 0-linked, or exclusively on 0-linked, chains of glycoproteins and also glycosphingolipids (for a review see reference 9). HTLVIIIB was preincubated with hybridoma supernatants and then used to inoculate MT4 cells. Those hybridoma supernatants which reduced HIV antigen concentration in the culture supernatants after 4 days of culture were then dialyzed against PBS to remove any preservative and retested for HIV neutralization (Fig. 1). The following MAbs reduced HIV antigen concentration in the culture supernatants to background levels: TKH2, BM1, AH21, and AH16 (AH16 having similar but broader specificity than AH21). Cells from these cultures were analyzed for HIV DNA by polymerase chain reaction after 4 days of culture. All cultures with HIV antigen-negative supernatants (TKH2, BM1, AH21, AH16, and the uninfected control) were also negative for HIV DNA, and cultures with antigen-positive supernatants were also positive for HIV DNA. Purified antibodies of hybridoma culture supernatants of AH21, BM1, TKH2, and B72.3 (having the same specificity as TKH2) showed a concentration-dependent inhibition of
0.61 1.02 1.28 Microgram MAb per TCID60
0 0.016 0.044 0.133 0.400 Microgram MAb per TCID5O
Percent Inhibition by MAb B72.3
Percent inhibition by MAb TKH2
2 0.2 0.02 Microgram MAb per TCID60
0.2 0.003 0.013 0.05 Microgram MAb per TCID60
FIG. 2. Concentration-dependent inhibition of HIV infection in MT4 cells. HTLVIIIB was preincubated with various concentrations of purified carbohydrate-specific MAb. HIV antigen in culture supernatants was measured by ELISA at day 4 and expressed relative to cultures inoculated with untreated virus (percent inhibition = 0). MAb concentration was expressed relative to virus inoculum (TCID50). Means of quadruplicate experiments are shown.
VOL. 64, 1990
HIV INHIBITION BY CARBOHYDRATE-SPECIFIC MAbs
Percent inhibition of control
Percent inhibition of control
Monoclonal antibody 20
Microgram MAb per TCID50 FIG. 3. Inhibition of HIV infection in MT4 cells. A fresh HIV-1 patient isolate (SSI-002) was preincubated with purified carbohydrate-specific MAb. HIV antigen in culture supernatant was measured by ELISA at day 4 and expressed relative to cultures inoculated with untreated virus (percent inhibition = 0). MAb concentration was expressed relative to virus inoculum (TCID50). Means of quadruplicate experiments are shown. infection (Fig. 2). The inhibitory antibody concentration was defined as the antibody concentration resulting in an 80% reduction of HIV antigen expression after 4 days of culture, as described for neutralizing MAb titers (21), and expressed relative to infectious dose. The inhibitory concentrations were interpolated to be as follows: B72.3, 0.18 ptg/TCID50; AH21, 0.32 pug/TCID50; BM1, 1.16 tLg/TCID50; and TKH2, 1.6 xug/TCID50. The MAbs inhibited infection with both HIV-1 reference strain HTLVIIIB and isolate SSI-002 (Fig. 3) when the virus preparation was preincubated with MAb before inoculation onto MT4 cells. No inhibition of HIV infection was found when the cells were preincubated with MAb, washed, and then inoculated with untreated virus. In order to compare the inhibitory effect of the MAbs on HIV infection in different cell systems, cells of the MT4 cell line, PHA-stimulated PMC, and selected T4 cells from a normal donor were used as target cells for infection with HTLVIIIB preincubated with the MAbs AH21, TKH2, B72.3, and BM1. Infection was reduced to the same extent in the three cell systems with all four antibodies (Fig. 4). The MAbs found to inhibit the infectivity of cell-free virus were also found to inhibit formation of syncytia when HTLVIIIB-infected cells were preincubated with antibody prior to coculture with uninfected CEM cells (Table 2). Compared with cultures without antibody, cell doubling time and percentage of live cells were unaffected by antibody. For example, no toxicity of MAbs was found when MT4 cells were incubated with 20 ,ug of MAb per ml (the highest concentration used in infectivity assays). The number of live cells per microliter was estimated by trypan blue
FIG. 4. Inhibition of HIV infection in different cells. HTLV1J1B was preincubated with purified carbohydrate-specific MAb (50% inhibitory concentration in MT4 cells) and used to infect PMC from a normal donor (-), T4 lymphocytes selected by "panning" from a normal donor (S), or cells from a T-cell line (MT4) (0L). HIV antigen in culture supernatants was measured by ELISA at day 4 and expressed relative to cultures inoculated with untreated virus (percent inhibition = 0). Means of quadruplicate experiments are shown.
exclusion (mean + standard error of the mean of quadruplicate determinations). On day 0 the value was 500 + 41 and on day 4 they were as follows: AH21, 920 + 203; BM1, 1,340 ± 165; B72.3, 940 + 112; TKH2, 1,360 ± 180; PBS, 1,200 + 133. The percentage of live cells was greater than 80% in all cultures. In infectivity experiments, the live-cell count at day 4 and especially at day 7 in cultures infected with HIV, which had been preincubated with one of the neutralizing MAbs, was in all cases higher than in cultures infected with untreated HIV. FACS analysis (Table 3) showed that the primary target cells, MT4, used in inhibition experiments did not stain with the inhibitory MAbs at all (BM1 and AH16) or only to a low degree (B72.3, 13%). After infection, the percentage of cells stained by B72.3 increased significantly. Background staining of MT4 cells increased after infection from 4 to 25%, probably because of extensive cell death caused by HIV, and the percentages of cells stained by BM1 (28%) and AH16 (26%) were thus not significantly increased after 4 days of infection. The primary virus sources were, however, H9 cells permissively infected with HTLVIIIB. These cells, TABLE 2. Reduction of syncytial activity by carbohydratespecific MAbs after preincubation of 10,000 infected H9 cells with 7.5 ,g of antibody (including the negative control anti-CD8)' Syncytia Antibody
None Anti-CD8 B72.3 (same as TKH2) AH16 (same as AH21) BM1
41 46 16 12
No. per 100,000 live cells
20 11 2
a All syncytia (giant cells with more than 5 nuclei) in each culture were counted after 24 h of coculture with 100,000 uninfected CEM cells.
HANSEN ET AL.
TABLE 3. Staining of various cells of lymphocyte lineage by carbohydrate-specific MAbs
% Positive cells in FACS analysis Primary antibody
12.0 96.2 14.0 26.5 18.5 67.5
25.4 3.7 None 96.5 8.1 Anti-gp120 36.6 99.9 Leu3a 27.7 4.5 BM1 26.2 4.4 AH16 (same as AH21) 68.3 13.4 B72.3 (same as TKH2) a MT4/3B, MT4 cells infected with HTLVIB at day 4. b H9/3B, H9 cells permissively infected with HTLVIIB.
which were also used in syncytium experiments, stained with all three MAbs above background level. Control staining with anti-gp120 and anti-CD4 (anti-Leu3a) showed a down regulation of CD4 expression concomitant with the appearance of membrane-bound gp120 after HIV infection. Radioimmunoprecipitation of a 1251I-labeled HIV lysate with MAbs showed that the neutralizing MAbs AH16 (Fig. SB, lane 1), TKH2 (Fig. SB, lane 2), and BM1 (Fig. 6, lane 2) reacted specifically with the major viral envelope glycoprotein gp120. Viral proteins of 30 to 60 kilodaltons (kDa) were unspecifically precipitated by negative controls (normal serum and nonneutralizing MAbs SH1 and FH7) as well as by AH16, TKH2, and BM1. An additional viral protein of approximately 70 to 80 kDa was precipitated by serum from an HIV-infected patient, AH16, TKH2, and BM1, but relatively less precipitated with negative controls. DISCUSSION Carbohydrate residues on the HIV envelope glycoprotein gp120 have been implicated in adhesion to CD4, initiating HIV infection of lymphocytes. Thus, deglycosylation of gp120 abrogates binding to the CD4 receptor (29), and B
FIG. 5. Radioimmunoprecipitation, using protein A-Sepharose, of 125I-labeled HIV lysate with serum from a HIV-1 infected patient (Al) or carbohydrate-specific MAbs of IgG class (Bi, AH16; B2, TKH2). Controls were normal donor serum (A2) or nonneutralizing carbohydrate specific-MAb SH1 (B3) of IgG class. Al and A2 are from the same 8% polyacrylamide gel electrophoresis gel and Bi, B2, and B3 are from the same 10% polyacrylamide gel electrophoresis gel.
FIG. 6. Radioimmunoprecipitation, using goat anti-mouse IgM bound to protein A-Sepharose, of '25I-labeled HIV lysate with serum from a HIV-1 infected patient (lane 1), neutralizing carbohydrate-specific MAb BM1 of IgM class (lane 2) or nonneutralizing carbohydrate-specific MAb FH7 of IgM class (lane 3). All lanes are from the same 10% polyacrylamide gel electrophoresis gel.
inhibition of Golgi-mediated glycosylation in HIV-infected cells reduces the infectivity of HIV produced (16, 31). A similar antiviral effect of glycosylation inhibition has been found in vivo in a murine system in which Rauscher leukemia virus was used (42). Recently, the core of N-linked glycosylation of gp120 of recombinant HIV has been characterized (30). In this study, gp120 recombinant protein was produced in Chinese hamster ovary cells, which should produce a different glycosylation pattern of recombinant gp120 from that of gp120 proteins propagated in human T lymphocytes. In particular, the peripheral pattern would be expected to vary. Although the mannose core of N-linked carbohydrates exhibits structural variations as a result of varying exoglycosidase and glucosaminyltransferase modification after the initial addition of Glc3Man9GlcNAc2 (where Glc is glucose, Man is mannose, and GlcNAc is N-acetylglucosamine) to asparagine residues of the protein core (for a review, see reference 43), it is generally the peripheral glycosylation, i.e., the poly-N-acetyllactosamine "antennae," that shows varying structure and immunogenicity. Distinct peripheral glycosylation patterns are thus a characteristic of cell type, cell maturation, and differentiation processes, as well as of oncogenic transformation (12, 14, 17). Many of these peripheral carbohydrate structures elicit strong immune responses in animals and humans. In the present study we have used a panel of murine MAbs defining peripheral carbohydrate structures (generally 2 to 5 residues) which may be found on poly-N-acetyllactosamine structures of N- and 0-linked glycoproteins and glycosphingolipids, as well as on structures restricted to 0-linked glycoproteins (Table 1). MAb BM1, directed to Ley and previously found to be expressed on HIV-infected lymphocytes (3, 39), inhibited formation of syncytia between infected and uninfected cells and also the infectivity of cellfree virus. This inhibition was observed only on preincubation of MAb with virus, but not of MAb with cells, before inoculation. Furthermore, BM1 precipitated gp120 in an 125I-labeled HIV lysate. Two MAbs, AH21 (IgM) and AH16 (IgG3), with specificity for blood group A type 1 chain antigen (1), also showed inhibition, whereas MAbs directed to other A-antigen types did not. This inhibition was probably a result of interaction with the viral envelope since AH16 did not bind to target
VOL. 64, 1990
HIV INHIBITION BY CARBOHYDRATE-SPECIFIC MAbs
cells prior to infection and since AH16 precipitated the major viral envelope glycoprotein gpl20. The finding that MAbs TKH2 and B72.3, directed to the sialyl-Tn antigen, inhibited infection and syncytium formation and also precipitated gpl20, suggests that 0-linked glycans may also exist on the HIV envelope, as has been found in other enveloped viruses (36) but not previously in HIV. The relatively stronger precipitation of gpl20 produced with AH16 than with THK2 could be caused by differences in affinity; this is probably also the reason for the different potency of HIV neutralization by TKH2 and B72.3. From previous studies (18, 30), gpl20 would, however, be expected to contain much more N-linked than 0-linked glycans, and the 0-linked glycans may possibly be present only on some gpl20 molecules. This would present more epitopes to AH16 than to TKH2. In addition to gpl20, a viral protein of approximately 80 kDa was also precipitated by AH16, TKH2, and patient serum. This band was also faintly present after immunoprecipitation with normal serum and nonneutralizing MAb SH1, indicating some degree of unspecific binding. The band at 80 kDa was relatively stronger than the gpl20 band when precipitated with patient serum but relatively weaker than the gpl20 band when precipitated with carbohydrate-specific MAbs TKH2 and AH16. We therefore believe that the band at 80 kDa may represent partly glycosylated gpl20, as previously suggested (4). The glycosylation of viral proteins is believed to be performed by the host cell gene-encoded glycosyltransferases. Therefore, it is generally expected that the host cell and proteins of viruses propagated therein show similar glycosylation patterns. The three carbohydrate neutralization epitopes (Ley, A1, and sialyl-Tn) found in this study are not normally found on human lymphocytes. We found all the epitopes to be expressed in chronically HIV-infected lymphocytes, which corresponds well with previous studies of Ley expression (3, 39), and for at least one epitope (sialylTn), we could demonstrate an increased expression after HIV infection. We therefore suggest that viral infection may induce expression of abnormal glycosyltransferase genes in host cells, possibly by the action of an HIV-specific transactivating factor, and that this may result in viral incorporation of carbohydrate structures not normally found in the host cell. Furthermore, the final glycan structure of a glycoprotein is a result of both exoglycosidase and glycosyltransferase activity as well as the primary structure of the core protein, which in the case of viral glycoproteins could in itself result in novel glycosylation patterns. This intriguing problem may only be solved by detailed chemical and biosynthetic studies of HIV protein glycosylation. HIV-neutralizing antibodies are found in HIV-infected patients and have also been produced in vitro by using selected synthetic peptides from gpl20 and gp4l (8, 25, 44). These do not seem to protect against disease progression and are type specific. "Escape" mutants of HIV, containing mutations in the env gene, have been shown to arise in vitro, whereby the virus is able to escape the effect of formerly neutralizing antibodies (45). The anti-HIV reactivity of antisera against equine infectious anemia virus is directed against a carbohydrate part of the HIV envelope (32), but HIV neutralization by carbohydrate-specific antibodies has not been described earlier. Carbohydrate epitopes may not be expected to be influenced as readily by mutations in the genome coding for the peptide part of the HIV envelope. Thus, our results indicate that
viral glycans may be possible targets for antiviral immuno-
therapy or vaccine development or both. ACKNOWLEDGMENTS This study was supported by grant 12-7928 and 12-7941 from The Danish Medical Research Council, by grant 03.2821:42 from The Danish Insurance Association, by National Cancer Institute Outstanding Investigator grant CA42505 (to S.-I.H.), and by funds from The Biomembrane Institute. E.D. and H.C. were supported by Ingeborg og Leo Dannin Fonden, Vera og Carl Johan Michaelsons Fond, Denmark; L.S.T. was supported by Sygekassernes Helsefond, Denmark. We wish to thank Henning Heldbjerg and Berit Hansen for expert technical assistance and Masakazu Adachi for his support. LITERATURE CITED 1. Abe, K., S. B. Levery, and S. Hakomori. 1984. The antibody specific to type 1 chain blood group A determinant. J. Immunol. 132:1951-1954. 2. Abe, K., J. M. McKibbin, and S.-I. Hakomori. 1983. The monoclonal antibody directed to difucosylated type 2 chain (Fuc 1-2Galpl-4[Fuc 1-3]GlcNAc; Y determinant). J. Biol. Chem. 258:11793-11797. 3. Adachi, M., M. Hayami, N. Kashiwagi, T. Mizuta, Y. Ohta, M. J. Gill, D. S. Matheson, T. Tamaoki, C. Shiozawa, and S. Hakomori. 1988. Expression of LeY antigen in human immunodeficiency virus-infected human T cell lines and in peripheral lymphocytes with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC). J. Exp. Med. 167: 323-331. 4. Allan, J. S., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A. Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:10911093. 5. Andrews, P. W., E. Gonczol, B. A. Fenderson, E. H. Holmes, G. O'Malley, S. Hakomori, and S. Plotkin. 1989. Human cytomegalovirus induces stage-specific embryonic antigen 1 in differentiating human teratocarcinoma cells and fibroblasts. J. Exp. Med. 169:1347-1360. 6. Bahraoui, E., B. Clerget-Raslain, F. Chapuis, R. Olivier, C. Parravicini, M. Yagello, L. Montagnier, and J.-C. Gluckman. 1988. A molecular mechanism of inhibition of HIV-1 binding to CD4+ cells by monoclonal antibodies to gpllO. AIDS 2:165169. 7. Boyum, A. 1968. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. Suppl. 97:1-108. 8. Chanh, T. C., G. R. Dreesman, P. Kanda, G. P. Linette, J. T. Sparrow, D. D. Ho, and R. C. Kennedy. 1986. Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J. 5:3065-3071. 9. Clausen, H., and S. Hakomori. 1989. ABH and related histoblood group antigens: immunochemical differences in carrier isotypes and their distribution. Vox Sang. 56:1-20. 10. Clausen, H., S. B. Levery, E. Nudelman, S. Tsuchiya, and S. Hakomori. 1985. Repetitive A epitope (type 3 chain A) defined by blood group A-I specific monoclonal antibody TH-1: chemical basis of qualitative Al and A2 distinction. Proc. Natl. Acad. Sci. USA 82:1199-1203. 11. Clausen, H., M. R. Stroud, J. Parker, G. Springer, and S. Hakomori. 1988. Monoclonal antibodies directed to the blood group A associated structure, galactosyl-A: specificity and relation to the Thomsen-Friedenreich antigen. Mol. Immunol. 25:199-204. 12. Feizi, T., and R. A. Childs. 1985. Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumorassociated antigens and components of receptor systems. Trends Biochem. Sci. 10:24-27. 13. Foley, G. E., H. Lazarus, S. Farber, B. G. Uzman, B. A. Boone, and R. E. McCarthy. 1965. Continous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18:522-529. 14. Fukuda, M. 1985. Cell surface glycoconjugates as onco-differ-
HANSEN ET AL.
entiation markers in hematopoietic cells. Biochim. Biophys. Acta 780:119-150. Fukushi, Y., E. Nudelman, S. B. Levery, H. Rauvala, and S. Hakomori. 1984. Novel fucolipids accumulating in human cancer. III. A hybridoma antibody (FH6) defining a human cancerassociated difucoganglioside (VI3NeuAcV3III3Fuc2nLc6). J. Biol. Chem. 259:10511-10517. Gruters, R. A., J. J. Neefjes, M. Tersmette, R. E. Y. Goede, A. Tuip, H. G. Huismann, F. Miedema, and H. L. Ploegh. 1987. Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase. Nature (London) 330:74-77. Hakomori, S. 1985. Aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives. Cancer Res. 45:2405-2414. Hansen, J.-E., C. M. Nielsen, C. Nielsen, P. Heegaard, L. R. Mathiesen, and J. 0. Nielsen. 1989. Correlation between carbohydrate structures on the envelope glycoprotein gpl20 of HIV-1 and HIV-2 and syncytium inhibition with lectins. AIDS 3: 635-641. Harada, S., Y. Koyanagi, and N. Yamamoto. 1985. Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay. Science 229:563-566.
20. Hirohashi, S., H. Clausen, T. Yamada, Y. Shimosato, and S. Hakomori. 1985. Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group 0 or B individuals: its identification as Tn antigen. Proc. Natl. Acad. Sci. USA 82:7039-7043. 21. Kinney Thomas, E., J. N. Weber, J. McClure, P. R. Clapham, M. C. Singhal, M. K. Shriver, and R. A. Weiss. 1988. Neutralizing monoclonal antibodies to the AIDS virus. AIDS 2:25-29. 22. Kjeldsen, T., H. Clausen, S. Hirohashi, T. Ogawa, H. lijima, and S. Hakomori. 1988. Preparation and characterization of monoclonal antibodies directed to the tumor-associated 0-linked sialosyl-2-6-N-acetylgalactosaminyl (sialosyl-Tn) epitope. Cancer Res. 48:2214-2220. 23. Kozarsky, K., M. Penman, L. Basiripour, W. Haseltine, J. Sodroski, and M. Krieger. 1989. Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein. J. Acquired Immune Defic. Syndr. 2:163-169. 24. Kumarasamy, R., and H. A. Blough. 1985. Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. I. Surface labeling and serial lectin-binding studies of Asnlinked oligosaccharides of glycoprotein gC. Arch. Biochem. Biophys. 236:593-602. 25. Lasky, L. A., J. E. Groopman, C. W. Fennie, P. M. Benz, D. J. Capon, D. J. Dowbenko, G. R. Nakamura, W. N. Nunes, M. E. Renz, and P. W. Berman. 1986. Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science 233:209-212. 26. Lasky, L. A., G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodeficiency virus type 1 gpl20 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. 27. Lifson, J., S. Coutr6, E. Huang, and E. Engleman. 1986. Role of envelope glycoprotein carbohydrate in human serum immunodeficiency virus (HIV) infectivity and virus-induced cell fusion. J. Exp. Med. 164:2101-2106. 28. Linsley, P. S., J. A. Ledbetter, E. Kinney-Thomas, and S.-L. Hu. 1988. Effects of anti-gpl20 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1. J. Virol. 62:3695-3702. 29. Matthews, T. J., K. J. Weinhold, H. K. Lyerly, A. J. Langlios, H. Wigzell, and D. P. Bolognesi. 1987. Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gpl20 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. Proc. Natl. Acad. Sci. USA 84:54245428. 30. Mizuochi, T., M. W. Spellman, M. Larkin, J. Solomon, L. J. Basa, and T. Feizi. 1988. Structural characterization by chromatographic profiling of the oligosaccharides of human immunodeficiency virus (HIV) recombinant envelope glycoprotein
gpl20 produced in Chinese hamster ovary cells. Biomed. Chromatogr. 2:260-266. Montefiori, D. C., W. E. Robinson, and W. M. Mitchell. 1988. Role of N-glycosylation in pathogenesis of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 85:9248-9252. Montelaro, R. C., W. G. Robey, M. D. West, C. J. Issel, and P. J. Fischinger. 1988. Characterization of the serological crossreactivity between glycoproteins of the human immunodeficiency virus and equine infectious anaemia virus. J. Gen. Virol. 69:1711-1717. Nielsen, C. M., I. C. Bygbjerg, and B. F. Vestergaard. 1987. Detection of HIV antigens in eluates form whole blood collected on filter paper. Lancet i:566-567. Nudelman, E., Y. Fukushi, S. B. Levery, T. Higuchi, and S. Hakomori. 1986. Novel fucolipids of human adenocarcinoma: disialosyl Le-a antigen (III4FucIII6NeuAcIV3NeuACLc4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure. J. Biol. Chem. 261:5487-5495. Nuti, M., Y. A. Teramoto, R. Mariani-Constantini, P. H. Hand, D. Colcher, and J. Schlom. 1982. A monoclonal antibody (B72.3) defines patterns of a novel tumor-associated antigen in human mammary carcinoma cell populations. Int. J. Cancer 29:539546. Olofsson, S., J. Blomberg, and E. Lycke. 1981. 0-glycosidic carbohydrate-peptide linkages of herpes simplex virus glycoproteins. Arch. Virol. 70:321-330. Ou, C.-Y., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky, J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman. 1988. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononulear cells. Science 239:295-297. Park, M. B., R. Oriol, S. Nakata, P. I. Terasaki, R. Ford, and D. Bernoco. 1979. ABH and Lewis antigens on lymphocytes screening of pregnant womens sera with the B cell cytotoxicity test. Transplant. Proc. 11:1947-1949. Pincus, S. H., K. Wehrly, and B. Chesebro. 1989. Treatment of HIV tissue culture infection with monoclonal antibody-ricin A chain conjugates. J. Immunol. 142:3070-3075. Popovic, M., M. G. Sarngadharan, E. Read, and R. C. Gallo. 1984. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500. Ray, E. K., and H. A. Blough. 1978. The effect of herpes virus infection and 2-deoxy-D-glucose on glycosphingolipids in BHK21 cells. Virology 88:118-127. Ruprecht, R. M., S. Mullaney, J. Andersen, and R. Bronson. 1989. In vivo analysis of castanospemine, a candidate antiviral agent. J. Acquired Immune Defic. Syndr. 2:149-157. Snider, M. D. 1984. Biosynthesis of glycoproteins: formation of N-linked oligosaccharides, p 163-198. In V. Ginsburg and P. W. Robbins (ed.), Biology of carbohydrates, vol. 2. John Wiley & Sons, Inc., New York. Weber, J. N., P. R. Clapham, R. A. Weiss, D. Parker, C. Roberts, J. Duncan, I. Weller, C. Carne, R. S. Tedder, A. J. Pinching, and R. Cheingsong-Popov. 1989. Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis. Lancet i:119-122. Weiss, R. A., P. R. Clapham, M. 0. McClure, J. A. McKeating, A. McKnight, A. G. Dalgleish, Q. J. Sattentau, and J. N. Weber. 1988. Human immunodeficiency viruses: neutralization and receptors. J. Acquired Immune Defic. Syndr. 1:536-541. Wysocki, L. J., and V. L. Sato. 1978. "Panning" for lymphocytes: a method for cell selection. Proc. Natl. Acad. Sci. USA 75:2844-2848. Young, W. W., Jr., H. S. Johnson, Y. Tamura, K.-A. Karlsson, G. Larson, J. M. R. Parker, D. P. Khare, U. Spohr, 0. Baker, 0. Hindsgaul, and R. U. Lemieux. 1983. Characterization of monoclonal antibodies specific for the Lewis A human blood group determinant. Fed. Proc. 42:3209. Young, W. W., Jr., J. Portoukalian, and S. Hakomori. 1981. Two monoclonal anticarbohydrate antibodies directed to glycosphingolipids with a lacto-N-glycosyl type II chain. J. Biol. Chem. 256:10967-10972.