Vol. 66, No. 4

JOURNAL OF VIROLOGY, Apr. 1992, p. 2473-2483

0022-538X/92/042473-11$02.00/0 Copyright © 1992, American Society for Microbiology

Influence of Carbohydrate Moieties on the Immunogenicity of Human Immunodeficiency Virus Type 1 Recombinant gpl60 ABDELAZIZ BENJOUAD,lt* JEAN-CLAUDE GLUCKMAN,1 HERVE ROCHAT,2 LUC MONTAGNIER,3 AND ELMOSTAFA BAHRAOUI2 Laboratoire de Biologie et Genetique des Pathologies Immunitaires, Centre National de la Recherche Scientifique URA 1463, CERVI H6pital de la Pitie-Salpetnrie, 83 Boulevard de l'H6pital, 75651 Paris Cede-x 13,1 Unite d'oncologie virale, Institut Pasteur, 75015 Paris,3 and Centre National de la Recherche Scientifique URA 1455, Laboratoire de Biochimie, Faculte de Medecine Nord, 13326 Marseille Cedex 15,2 France Received 26 September 1991/Accepted 16 December 1991

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gpl60 and gpl20) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gpl60 or against gpl60 deglycosylated by either endo F-N glycanase, neuraminidase, or oa-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gpl60 (rgpl60) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gpl60 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gpl60 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gpl60 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gpl20. Rabbits immunized with desialylated rgpl60 generated antibodies which recognized not only rgpl60 from HIV-1 but also rgpl40 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgpl60 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gpl60 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against a-mannosidase-treated gpl60 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gpl60 can modulate the specificity and the protective efficiency of the antibody response to the molecule.

moieties present on the mature envelope glycoproteins does not abolish binding to CD4 or HIV-1 infectivity. Glycans may, however, play a critical role in the humoral immune response to viruses (18, 38, 50) by masking antibody binding to neutralizing sites or by directing the antibody response to nonneutralizing sites (1). In order to investigate the role of carbohydrate moieties in modulation of the humoral immune response, we analyzed the antibody response obtained for rabbits that had been immunized against either glycosylated or deglycosylated recombinant envelope glycoprotein precursor (gp160) of HIV-1. In the latter case, gpl60 was treated with neuraminidase to remove sialic acid, with ca-1-mannosidase to cleave external mannose residues, or with endo F-N glycanase, which efficiently cleaves both N-linked high-mannose and complex glycans.

The external envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) is responsible for virus binding to the cell surface CD4 receptor (4, 22, 46), while transmembrane glycoprotein gp4l is involved in fusion between the virus and host cell membranes (23). Both gpl20 and gp4l are involved in the induction of syncytium formation between infected and uninfected CD4-positive cells (27, 54). The gpl20 envelope glycoprotein is heavily glycosylated (8, 9, 28), glycans representing about 50% of its total molecular weight, and N-glycosylation sites are equally distributed between the variable and conserved regions of the molecule. HIV envelope glycoproteins represent the major target of neutralizing antibodies (14, 25, 41, 45), as recently confirmed by Berman et al. (3) and Girard et al. (12). The role of N-linked glycans in the HIV replicative cycle has been demonstrated by several studies. Glycans have been shown to be important for the proper intracellular processing, targeting, and function of gp120 and virus particles (15, 39, 40, 43). Virus produced in the presence of glucosidase inhibitors presents markedly reduced infectivity and cytopathogenicity (15, 43). But our group (8, 9), using enzymatically deglycosylated recombinant gpl20 and gpl60 or virus particles, has shown that removal of carbohydrate

*

MATERIALS AND METHODS Soluble recombinant gpl60 and gpl40. Recombinant soluble gpl60 of HIV-1 LAV-LAI (56), previously named LAVBRU (55), and gpl40 of HIV-2 LAV-ROD (16) were obtained from Transgene S.A. (Strasbourg, France). Briefly, the env coding sequence of gpl60 is derived from the HIV-1 LAV envelope precursor protein with deletion of the potential transmembrane segment (Ile-689 to Val-710) and in which the potential coding sequence of the cleavage site was mutated to prevent processing of gpl60 to gp120 and gp4l. Recombinant vaccinia virus-infected BHK cells produced gp160, which was then purified from the supernatant by

Corresponding author.

t Present address: Laboratoire de Biochimie, Faculte de Medecine Nord, Bd. Pierre Dramard, 13015 Marseille, France. 2473

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BENJOUAD ET AL.

J. VIROL. TABLE 1. Amino acid sequences of the synthetic peptidesa

Peptide

Peptide

P1 P3 P27 P44 P4 P5 P41 P28 P29 P30 P22 P21 P8 P50 P48 P47 P38 P56 P39 P40

Amino

Amino acid sequence

~~acids 33-55 115-131 166-190

192-201 193-212 221-234 252-266 268-280 308-328 323-336 418-461 487-534 487-508 503-516 529-549 546-564 561-586 578-596 583-610 622-641

KLWVTVYYG VWKEATTTLFCA SLKPCVKLTPLCVSLKC ISTSIRGKVQEYAFFYXLDIIPID DTTSYTLTSC

TTSYTLTSCNTSVITQACPK HYCAPAGFAILKCN CTHGIRPVV8TQLLL GSLAEEEVVIRSA TRKSIRIQRGPGRAFVTIGKI VTIGKIGNMRQAHC TITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRD ELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGST ELYRYKVVKIEPLGVAPTKAKR PTKARBRVVQREKR GAAGSTMGARSITLTVQARQL ARQLLSGIVQQQNNLLRAI LRAIEAQQHLLQLTVWGIKQLQARIL IKQLQARILAVERYLKDQQ RILAVERYLKDQQLLGIWGCSGKLICT KSLEQIWNNMTWMEWDREIN

% Homology with HIV-2

65 82 0 10 55 64 26 30 17 31 40 52 63 41 56 60 57 57 46 45

a The percentage of amino acid homology between HIV-1 LAV-LAI and HIV-2 LAV-ROD for the corresponding envelope glycoprotein regions is indicated. The one-letter amino acid code is used.

precipitation with ammonium sulfate and ethanol, followed by reverse-phase high-pressure liquid chromatography

(HPLC) (21).

gpl40 of HIV-2 (16) was produced by a similar protocol and purified from the cell supernatant by adsorption on heparin-Ultrogel followed by gel filtration on Sephacryl S 300 and anion-exchange HPLC. Analytical deglycosylation of gpl60. Three glycosidases were used: endo F-N glycanase (Boehringer Mannheim Biochemicals, Mannheim, Germany), which contains both endo F and N glycanase activities; neuraminidase; and ot-1-mannosidase (Sigma). Deglycosylation was performed as described before (8). Rabbit immunization with gpl60 or its deglycosylated derivatives. Nontreated gpl60 and its different deglycosylated forms were kept frozen at -20°C as 200-pLg aliquots until injected into the animals. Two New Zealand White rabbits were immunized with each immunogen as follows: 200 p,g (in 1 ml of phosphate-buffered saline [PBS] buffer) of each preparation mixed with an equal volume of complete Freund's adjuvant was injected intradermally on day 0. This procedure was repeated subcutaneously with incomplete Freund's adjuvant on days 30, 60, 90, and 120. Animals were bled for serum 1 week after each injection. Sera showing the highest immunoreactivity were further analyzed. RIA. Radiolabeling of both recombinant gpl60 and gpl40 with 125I was performed as described before (8). [125I]gp160 (50 pl, 8 x 10 to 1 x 105 cpm) was incubated with 50 pl of various serum dilutions and incubated for 2 h at 37°C. The complexes formed between [125I]gpl6O and anti-gpl60 antibodies were immunoprecipitated with 100 p1 of a suspension of protein A-Sepharose (1.5 g/40 ml) in PBS buffer (pH 7.4)-0.5% (wt/vol) bovine serum albumin (BSA) for 1 h at 37°C. After two washes with PBS buffer (pH 7.4)-0.5% (wt/vol) BSA-0.05% (wt/vol) NaN3-0.01% (vol/vol) Tween 20, bound radioactivity was counted in a gamma counter (Kontron Analytical, Zurich, Switzerland). Inhibition of binding of anti-gp160 antibodies to [1251]gpl60 by unlabeled protein was examined with the same procedure, except that

unlabeled protein was added in a competition assay. The radioimmunoassay (RIA) with [125I]gp140 was performed under the same conditions as above. ELISA. For the enzyme-linked immunosorbent assay (ELISA) for the detection of anti-gpl60 antibodies, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated for 2 h at 37°C with 50 ng of gpl60 or with 500 ng of a synthetic peptide per well in 50 RI of PBS, pH 7.4. After saturation with 400 pl1 of PBS (pH 7.4)-5% (wtlvol) casein for 1 h at 37°C and washing with PBS (pH 7.4)-0.01% (vol/vol) Tween 20, 50 p1 of serum dilutions was added and incubated for 2 h at 37°C. After the plates were washed, 50 p1 of swine anti-rabbit immunoglobulin G (IgG) coupled to peroxidase (1:5,000; Dakopatts) was incubated for 1 h at 37°C. After further washing, 100 pl of ortho-phenylenediamine in 0.03% H202 was added for 30 min at room temperature in darkness. The reaction was stopped by adding 50 pl of 4 N sulfuric acid, and the optical density was read at 492 and 620 nm to obtain the ratio. Wells coated with casein were used as negative controls. Synthetic peptides. Synthetic peptides spanning conserved and variable sequences of gpl60 were derived from the amino acid sequence of HIV-1 LAV-LAI (55, 56). The primary structures of these peptides and their homology with the corresponding regions of HIV-2 LAV-ROD (16) are shown in Table 1. CD4+ lymphoid cells. Cells of the Molt-4 (29), MT-4 (30), and CEM (10) lines were cultured at 37°C in RPMI 1640 medium (Flow Laboratories, Inc., Irvine, Scotland), supplemented with 10% fetal calf serum, 1% glutamine, and 1% antibiotics (GIBCO Laboratories, Paisley, Scotland), in a humidified atmosphere with 5% CO2. Assay for inhibition of binding of heat-inactivated HIV-1 to CD4-positive cells. One microgram of heat-inactivated HIV-1 LAV-LAI was incubated at 37°C with various serum dilutions in 50 p1 of PBS buffer containing 0.5% BSA and 0.05% NaN3. After 1 h of incubation, 5 x 105 CEM cells were added and incubated for 30 min at 37°C. After two washes, 25 pl of monoclonal antibody (MAb) 110-4 (Genetic Sys-

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tems, Seattle, Wash.) diluted 1:400 was added and incubated for 30 min at 4°C. After being washed, the cells were incubated for 30 min at 4°C with 25 ,u of a 1:25 dilution of anti-mouse IgG coupled to biotin (Amersham). After being washed again, the cells were then incubated in 25 ,u of a 1:800 dilution of streptavidin-phycoerythrin (Becton Dickinson and Co., Mountain View, Calif.). Cells were washed and stored in 500 ,l of 1% paraformaldehyde. A fluorescenceactivated cell sorter (FACS) analyzer (Becton Dickinson) was used to determine the intensity of cell membrane

fluorescence. HIV infection of MT4 cell cultures. Fifty microliters of different serum dilutions in RPMI 1640 medium were incubated in 96-well plates with 50 RI of HIV-1 LAV-LAI at a dilution equivalent to 25 to 50% tissue culture-infectious doses (TCID50). After 1 h at 37°C, 100 ,u of MT-4 cells (105 cells per ml) was added and cultured at 37°C in a CO2 incubator. Aliquots of cells and supematants were collected sequentially and analyzed to measure cell viability and virus production (assays in triplicate). Fresh medium was added to cultures to obtain a final volume of 200 RI. Colorimetric MTT assay. The colorimetric cell viability assay was performed as described before (48). The method is based on the cleavage of 3(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to formazan by mitochondrial enzymes present only in living cells (33). The formazan formed can be easily spectrophotometrically quantified at 540 nm. RT activity microassay. The reverse transcriptase (RT) activity microassay involves the direct measurement of RT activity in 50 ,u of culture supernatant. The procedure and the buffers are the same as those used by Schwartz et al. (48). Briefly, assays were carried out in triplicate: 10 RI of buffer A (0.5 M KCl, 50 mM dithiothreitol, 0.5% Triton X-100) and 40 RI of buffer B [10 ,u of 5 mM EGTA (ethylene glycol tetraacetic acid) in 0.5 M Tris-HCl (pH 7.8), 1 p1 of 0.5 M MgCl2, 3 pl of [3H]dTTP (Amersham), 10 ,u of poly(rA)oligo(dT) (5 OD units per ml), and 16 p1 of H20] were added to the wells. After 1 h at 37°C, 20 p1 of 12 mM Na4P207 in 60% trichloroacetic acid was added. Precipitates were filtered on Skatron filters, and radioactivity was counted in a liquid scintillation counter (Packard, Downers Grove, Ill.). Inhibition of syncytium formation between HIV-infected CEM cells and Molt-4 cells. CEM cells (104) chronically infected with HIV-1 LAV-LAI were incubated for 2 h with various serum dilutions. They were then cocultured with 4 x 104 uninfected Molt-4 cells at 37°C in 100 p1 of RPMI 1640 medium supplemented with 10% fetal calf serum, 1% glutamine, and 1% antibiotics in a humidified atmosphere with 5% CO2 in the wells (100 p1) of 96-well microtiter plates. After 18 h, the number of syncytia was counted. Preimmune sera from each rabbit were used as negative controls. As a positive control for the inhibition of cell fusion, anti-Leu3a (Becton Dickinson and Co.), an HIV-neutralizing anti-CD4 MAb, was used in parallel at 2.5 plg/ml in each experiment. RESULTS

Deglycosylation of gpl60. After treatment with the different glycosidases, deglycosylated gpl60 was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 shows that treatment of recombinant gpl60 with endo F-N glycanase leads to a reduction from 160 to 90 kDa (about 70 kDa) in the apparent molecular mass of the protein. Treatment with neuraminidase or mannosidase

a b 160 --

c

2475

d

-130 -90

FIG. 1. SDS-PAGE and autoradiography of U;5I]gpl60 after enzymatic treatment with different glycosidases. [1 I]gpl6O (11 ng) was incubated for 20 h at 37°C in the absence of glycosidase (lane a) or in the presence of a-l-mannosidase (lane b), neuraminidase (lane c), or endo F-N glycanase (lane d). Sizes are shown in kilodaltons.

decreased the molecular mass of gpl60 to 130 and 145 kDa, respectively (Fig. 1). Generation of antibodies against native and deglycosylated gpl60. Sera from rabbits that had been immunized with either native recombinant gpl60 or differently deglycosylated derivatives, after treatment with neuraminidase, a-mannosidase, or endo F-N glycanase, were tested by RIA for the detection of antibody binding to HIV-1 gpl60 and HIV-2 gpl40. Preimmune sera were used as controls. All immune sera tested presented high binding activity to radiolabeled gpl60, and the titers varied from 3 x 10-4 to 2 x 10-' (Fig. 2) (the titer is the serum dilution which yields 50% of maximum gpl60 binding). The specificity and avidity of the antibodies for gpl6O are shown in Fig. 3, in which different concentrations of unlabeled gpl60 were used to compete for antibody binding to [1251I]gpl60. The high avidity of antibody binding to gpl60 is indicated by a K05 value in the range of 10-9 M (Ko.5 being the concentration of unlabeled gpl6O inhibiting antibody binding to [125I]gpl6O by

50%). To further study the effect of carbohydrate chain moieties on the specificity of the antibodies elicited against native or differently deglycosylated gpl60, sera from all immunized rabbits were assayed for binding to radiolabeled recombinant gpl40 of HIV-2. No control preimmune sera had any significant immunoreactivity with [125I]gpl60 or [125I]gpl4O. Interestingly, only the sera from the rabbits that received desialylated gpl60 as an immunogen also exhibited strong

cross-reactivity with gpl40 (Fig. 4A), compared with the low or absent immunoreactivity noted with the sera from rabbits immunized with native or mannosidase- and endo F-N

glycanase-treated gpl60. Serum titers against gpl40 of antibodies produced against desialylated gpl60 varied from 2 x 10-3 to 5 x 10-4, and they were thus 10- to 30-fold lower than those noted against HIV-1 gpl60. In order to investigate whether this cross-reactivity was due to antiprotein or to anticarbohydrate antibodies, radiolabeled HIV-2 gpl40 was enzymatically deglycosylated by endo F-N glycanase treatment. We then compared the immunoreactivity of native and deglycosylated [ 25I]gpl4O as recognized by the cross-reactive antibodies produced against desialylated recombinant gpl60. The results depicted in Fig. 4B show that these antibodies recognize glycosylated and endo F-N glycanase-deglycosylated HIV-2 gpl40 to the same extent (serum titer between 2.5 x 10-3 and 7 x 10-4), which indicates that cross-reactivity is directed against the protein backbone rather than against the carbohydrate moieties of the molecules. Antigenicity of peptides mimicking different regions of

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BENJOUAD ET AL.

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IMMUNOGENICITY OF DEGLYCOSYLATED HIV-1 gpl60

VOL. 66, 1992

2477

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HIV-1 gpl60. Because it is possible that the carbohydrates side chains of gpl60 could influence the fine specificity of the elicited antibodies by masking or unmasking certain polypeptide regions, sera from rabbits immunized with native or deglycosylated gp160 were screened in an ELISA against a panel of synthetic peptides. Peptides were first chosen in order to analyze the immunogenicity of some critical regions of gpl60, including the principal neutralizing domain (P29) (14, 36, 41, 45, 52), the putative CD4 binding site (P22) (25), the potential fusogenic site (P21) (11), the immunodominant epitope in the sera of HIV-1-infected persons (P39) (13, 37, 47, 49, 53, 57), and some conserved regions of gpl60 (P1, P3, P4, P5, P8, P48, P47, P38, and P56). The other peptides were chosen in order to analyze the immunogenicity of different representative regions of gpl60. The amino acid sequence for each peptide (Table 1) was based on that of the HIV-1 LAV-LAI isolate (55, 56). Control preimmune sera recognized neither synthetic peptides nor recombinant gpl60 (result not shown). The results obtained (Fig. 5) show the following (i) Sera obtained after immunization with native gpl60

reacted only against peptides P29, P22, P21, P8, P56, and P39, and no significant reactivity against any of the conserved peptides P1, P3, P5, P48, P38, or P47 (average level of amino acid sequence conservation between HIV-1 and HIV-2, >50%) was noted. (ii) Only sera from rabbits immunized with desialylated gpl60 contained antibodies which could recognize peptide P41, in addition to the same spectrum of reactivity obtained with anti-native gpl60. (iii) Of the two rabbits immunized with endo F-N glycanase-treated gp160, one rabbit produced antibodies against the central region of V3 mimicked by peptide P29, while the other rabbit generated antibodies against the C-terminal region of the V3 region mimicked by peptide P30. (iv) No reactivity was noted against the V3 region in the sera of rabbits immunized with mannosidase-treated gpl60, but antibodies recognized the same other peptides as antinative gpl60. Inhibition of HIV-1 binding to CD4-positive cells by antibodies produced against native or deglycosylated gpl60. The results (Fig. 6) clearly show that all sera produced against

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native or deglycosylated gp160 could prevent virus binding to CD4-positive cells. The inhibiting antibody titer (the serum dilution required to inhibit virus binding by 50%) varied in the same range (1:3,000 to 1:14,000) as determined

by RIA against [1"I]gp160 (Fig. 2). No control preimmune sera inhibited the virus-CD4+ cell interaction. Neutralizing activity of anti-gpl6O antibodies. The neutralizing capacity of antibodies produced against native or

VOL. 66, 1992

IMMUNOGENICITY OF DEGLYCOSYLATED HIV-1 gpl6O

2479

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log of serum dilution FIG. 6. Inhibition of binding of inactivated HIV-1 to CD4-positive cells by antibodies produced against native gp160 (I and J) or gpl60 treated with neuraminidase (E and H), a-mannosidase (C and D), or endo F-N glycanase (A and B). Binding was determined by indirect immunofluorescence. Inhibition of binding (by various serum dilutions) was monitored by measuring fluorescence intensity with a FACS analyzer and expressed by reference to 100%, i.e., fluorescence intensity obtained in the presence of preimmune sera (0).

deglycosylated gp160 was analyzed by monitoring both cell viability with the MTT assay and virus replication by measuring RT activity in the supernatants of HIV-1-infected cultures. A second test involved using an HIV-induced syncytium inhibition assay. Rabbits immunized with native or desialylated gpl60 produced antibodies that neutralized HIV-1 infection of MT-4 cells with the equivalent of 25 TCID50. Sera used either at a 1:50 (data not shown) or at a 1:200 dilution (Fig. 7) were able to totally neutralize HIV-1 infection of MT-4 cells. Of the two rabbits that received completely deglycosylated gpl60, only one (panel A) had neutralizing antibodies, while the other (panel B) exhibited none. No neutralizing activity was observed in the sera of rabbits immunized with a-mannosidase-treated gp160. In the second assay, which assessed inhibition of syncytium formation (Fig. 8), only antibodies produced against native or desialylated gpl60 totally or partially inhibited syncytium formation in a dose-dependent manner. In contrast, no inhibition was obtained with sera from rabbits immunized with endo F-N glycanase- or a-mannosidasetreated gp160. Except for rabbit A, these results show a good correlation between the viral replication and syncytium

inhibition assays. It can be noted, however, that only sera (at a 1:20 dilution) produced by rabbits immunized with desialylated gpl60 totally prevented syncytium formation.

DISCUSSION The aim of this study was to determine whether carbohydrate moieties influence the immunogenicity of HIV-1 envelope glycoproteins, which represent the principal target of neutralizing antibodies. Despite the extensive glycosylation (8, 9, 28) of HIV-1 gpl60, the precise role of the carbohydrate moieties present on the mature molecules remains poorly understood, although several reports have stressed the importance of carbohydrate moieties for the function of retrovirus envelope proteins (1, 31, 32, 42, 43). For example, Skehel et al. (51) have shown that the addition of a glycosylation site at positions 63 to 65 in a variant of the 1968 Hong Kong influenza virus hemagglutinin prevented an MAb to the parent virus from recognizing the variant. Here, we have analyzed the influence of glycans on gpl60 immunogenicity in rabbits. It was found that antisera raised against the native or different deglycosylated derivative molecules recognized recombinant native gpl60 to the same

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Days in culture FIG. 7. Neutralization of HIV-1 infection by antibodies produced against native gpl60 (I and J) or gpl60 treated with neuraminidase (E and H), a-mannosidase (C and D), or endo F-N glycanase (A and B). Antibody neutralizing capacity was analyzed by monitoring, in the same test, both cell viability (MTT assay) and virus replication (RT activity). Sera were tested at a 1:200 dilution. Symbols: 0, cell viability; 0, RT activity.

extent. Only antibodies elicited by desialylated gp160 crossreacted with strong immunoreactivity against recombinant gp140 from HIV-2. One possible explanation is that, by demasking some regions, desialylation of recombinant gpl60 leads to the expression of new epitopes that are probably located within conserved regions between HIV-1 and HIV-2 envelope glycoproteins. A second interpretation is that after sialic acid removal, carbohydrate moieties might become immunogenic and lead to the production of antibodies specific to some oligosaccharide structures common to both

HIV-1 and HIV-2 envelope glycoproteins. These types of carbohydrate epitopes have recently been described by Hansen et al. (17) and Muller et al. (34), suggesting that carbohydrate epitopes should not be excluded in the development of an efficient vaccine against HIV-1, since these types of epitopes, not virally encoded, may not be expected to be influenced by the great variability of the virus genome. Our results indicate that glycosylated and deglycosylated HIV-2 gp140 is recognized to the same extent by antibodies produced against desialylated gp160.

IMMUNOGENICITY OF DEGLYCOSYLATED HIV-1

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100-

co

80-

c

60-

0)

40-

20

1:20

1:50 Serum dilution

1:200

FIG. 8. Inhibition of syncytium formation between HIV-1-infected and noninfected CD4-positive cells by antibodies produced against native gp160 (I and J) or gp160 treated with neuraminidase (E and H), a-mannosidase (C and D), or endo F-N glycanase (A and B). Inhibition of syncytium formation is expressed by reference to 100%, i.e., the number of syncytia obtained in the presence of preimmune sera, and 0%, i.e., the number of syncytia obtained in the presence of anti-CD4 Leu3a antibodies.

In order to further examine the effects of glycosylation on the humoral immune response, we analyzed the immunoreactivity of sera raised against native or deglycosylated gpl60 toward a panel of synthetic peptides corresponding to variable and conserved regions of HIV-1 gpl60. As shown previously (1, 5), mapping of these reactivities showed that serum reactivity to each peptide could increase, decrease, or remain unchanged. For example, only antibodies raised against desialylated gpl60 recognized peptide P41 (amino acids 252 to 262), mimicking a region without any putative glycosylation site but in proximity to three nonsialylated high-mannose or hybrid oligosaccharide types at asparagines 239, 246, and 267, according to the characterization of N-glycosylation sites reported by Leonard et al. (26). It is possible that removal of sialic acid from other glycans located at some distance causes an intrinsic modification in the conformation of the polypeptide backbone, so that antibodies against P41 could be produced only after immunization with desialylated gpl60. This immunoreactivity against P41, which mimicks a sequence that is weakly conserved (26%) between HIV-1 and HIV-2 envelope glycoproteins, however, is insufficient to explain the strong crossreactivity against HIV-2 gpl40 of antibodies produced against desialylated gpl60, as shown by the inability of this peptide to inhibit, in a competition assay, the interaction between [12I]gpl40 and antibodies against desialylated gpl60 (data not shown). The reactivity of the synthetic peptide P29 was analyzed in sera from rabbits immunized with glycosylated or deglycosylated gpl60 in order to analyze the influence of envelope glycoprotein carbohydrate moieties on the immunogenicity of the principal neutralizing determinant (14, 36, 41, 45, 52). The results presented here show that anti-P29 antibodies are present in rabbits immunized with native gpl60 (two of two), desialylated gpl60 (two of two), and endo F-N glycanasetreated gpl60 (one of two) but not in rabbits immunized with mannosidase-treated gpl60. Here again, the lack of P29 immunogenicity in the sera produced against mannosidase-

gpl6O

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treated gpl60 may be the consequence of an alteration of the V3 loop after this treatment or of conformational changes occurring in the protein by the deletion or introduction of new interactions after mannose residue removal, for example, the two mannosidase-sensitive high-mannose or hybrid glycans located at Asn-300 and Asn-337, which are located in proximity to the V3 loop by the conserved disulfide bridge between cysteines 301 and 336 (26). Finally, we have analyzed the capacity of these antibodies to neutralize HIV infection in vitro. All rabbits immunized with native or deglycosylated gpl60 possessed antibodies that inhibited the binding of HIV-1 particles or soluble gp160 (data not shown) to CD4, but this inhibiting effect was not correlated with the neutralizing capacity of the sera. Rabbits immunized with native or desialylated gp160 but not with a-mannosidase-treated gpl60 presented neutralizing antibodies, and only one of the two rabbits immunized with totally deglycosylated gpl60, the one whose serum contained antibodies against P29, produced neutralizing antibodies. The apparent discrepancy observed between the results of virus binding inhibition and those of HIV-1 neutralization could be due simply to differences in the sensitivity of the assays used; for example, the detection threshold of cytometry is estimated as 2,000 to 4,000 molecules per cell (7a), whereas very few particles are needed to infect a cell (28a). More interesting is the comparative analysis of epitope mapping among neutralizing and nonneutralizing sera, which clearly shows a good correlation between immunoreactivity against P29 and the capacity to neutralize HIV-1 infection. This is in good agreement with the finding of Javaherian et al. (19), Berman et al. (3), and Girard et al. (12). On the other hand, the presence of maternal antibodies against the V3 region seems to correlate with a reduced rate of mother-tochild transmission (6, 44). Moreover Emini et al. (7) have reported direct evidence of the protective effect of such antibodies in experimentally inoculated chimpanzees. However, it has been shown that antibodies against the V3 region generally neutralize virus replication and syncytium formation in a type-specific manner (14, 41). These antibodies do not inhibit binding of soluble gpl20 to CD4 (2), and they are able to block HIV infection when added to cells previously incubated with virus. Thus, antibodies seem to neutralize HIV infection by interfering with some step(s) subsequent to the attachment of virus to the target cells (35). Despite the hypervariability of the V3 loop, La Rosa et al. (24) found, by analyzing the amino acid sequence of this region from 245 different HIV-1 isolates, that the central sequence of this domain, GPGRAF, is conserved. Similar results were obtained by Wolfs et al. (58). Interestingly, when this region was used as an immunogen, broadly neutralizing antibodies were obtained (20). All in all, these results indicate that the carbohydrate moieties of HIV-1 gp160 can modulate the specificity of the antibody response, as demonstrated by the variation of the reactivity against the V3 loop region of antibodies raised against different forms of deglycosylated recombinant gpl60 and by the high cross-reactivity induced by desialylated recombinant gpl60. ACKNOWLEDGMENTS We thank Y. Henin, D. Guetard, A. LeHen, M. Yagello, and M. Alvitre for technical assistance. This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS) and the Fondation de L'Avenir, Paris, France.

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