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Bioconjugate Chem. 2006, 17, 493−500

493

Toward a Carbohydrate-Based HIV-1 Vaccine: Synthesis and Immunological Studies of Oligomannose-Containing Glycoconjugates Jiahong Ni, Haijing Song, Yadong Wang, Nicholas M. Stamatos, and Lai-Xi Wang* Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21201. Received September 20, 2005; Revised Manuscript Received January 19, 2006

Human antibody 2G12 is a broadly neutralizing antibody that exerts its anti-HIV activity by targeting a novel oligomannose cluster on HIV-1 gp120. It was previously demonstrated that synthetic oligomannose clusters could mimic the carbohydrate epitope of 2G12 and showed enhanced antigenicity (Wang L. X. et al. (2004) Chem. Biol. 11, 127). This paper describes the synthesis of oligomannose-containing glycoconjugates that include either a carrier protein or a universal T-helper epitope peptide to provide an effective immunogen. It was shown that the synthetic neoglycoconjugates containing oligomannose clusters could be recognized by the human antibody 2G12. Rabbit immunization studies revealed that only a small fraction of antibodies raised by the glycoconjugates was directed to the carbohydrate antigens, with the majority of the IgG type antibodies being directed to the linkers in the conjugates. The anti-sera showed weak cross-reactivity to HIV-1 gp120.

INTRODUCTION An effective protective vaccine against HIV-1 is believed to be the best hope to end the worldwide epidemic of HIV/AIDS. However, development of an effective HIV-1 vaccine has faced many challenges (1-3). To achieve maximal protection, both cellular immunity and humoral immunity are likely to be required for an effective HIV vaccine (4, 5). While cytotoxic T lymphocyte (CTL) response is important to reduce HIV-1 infection by destroying the infected cells (6), CTL alone cannot prevent or eliminate HIV infection. On the other hand, passive immunization with some broadly neutralizing monoclonal antibodies such as 2F5, 2G12, and 4E10 has repeatedly demonstrated in animal models that these neutralizing antibodies can provide sterilizing immunity when present at sufficient concentrations (7-9). Therefore, eliciting broadly neutralizing antibodies remains a high priority in designing a preventive HIV vaccine. Unfortunately, attempts to create immunogens capable of eliciting broadly neutralizing antibodies have been largely unsuccessful (4, 10, 11). This situation urges the exploration of new vaccine approaches. Among the several broadly neutralizing antibodies so far identified from AIDS patients (10-15), the human monoclonal antibody 2G12 is of particular interest, as it targets a unique carbohydrate antigenic structure on HIV-1 gp120 (15). Recent biochemical and structural studies suggest that the epitope of 2G12 consists of a novel cluster of oligomannose residues on the “silence face” of the HIV-1 envelope glycoprotein gp120 (16-19). Glycosylation with multiple high-mannose type N-glycans was found to be a common feature of the envelope glycoprotein gp120 of different HIV-1 strains so far analyzed (20-23). Such a high-density oligomannose clustering structure is rare for normal human glycoproteins. Thus a vaccine based on this unique antigenic structure may raise specific antibodies that would not be cross-reactive to normal human glycoproteins. Indeed, phase I clinical trials of the carbohydrate-specific antibody 2G12, together with another broadly neutralizing

antibody 2F5, showed that 2G12 are safe and well tolerated in humans (24, 25). The results suggest that targeting the oligomannose cluster on HIV-1 gp120 constitutes a feasible vaccine approach. As the first step toward this goal, several groups have performed 2G12-binding studies with structurally defined oligomannoses and/or related synthetic oligosaccharide clusters in order to define the antigenic structure (26-30). The binding studies indicated that the terminal ManR1,2Man unit was essential for 2G12 recognition but the ManR1,2Man unit alone was not sufficient for an effective binding to 2G12. In addition, it was shown that the full-size Man9 had the highest affinity to 2G12 among several natural high-mannose oligosaccharides, and the synthetic mannose tetrasaccharide corresponding to the D1 arm of Man9 showed comparable affinity to the antibody as that of the Man9 moiety (26). Using synthetic oligomannose clusters, we have further demonstrated that template-assembled oligomannose clusters are better mimics of the 2G12 epitope (27, 28). For example, the galactose-based tetravalent Man9cluster (Tetra-Man9) was 73-fold and 5000-fold more effective in binding to 2G12 than the monomeric Man9GlcNAc and Man6GlcNAc, respectively (27). These results suggest that the synthetic oligomannose cluster Tetra-Man9 may serve as a reasonable starting point for designing a carbohydrate-based HIV-1 vaccine. Since carbohydrate antigens alone are poorly immunogenic and are unable to raise T-cell dependent, IgGtype antibody responses, a conventional means to solve this problem is to conjugate the carbohydrate antigen to a strong T-helper epitope, e.g., a carrier protein, as exemplified by numerous studies on carbohydrate-based cancer vaccines (31). We describe in this paper the synthesis of conjugate vaccines in which Man9 and the oligomannose clusters are conjugated either to keyhole limpet hemocyanin (KLH) or to a universal T-helper epitope from tetanus toxoid (TT) (Figure 1). The glycoconjugates were used to immunize rabbits. The antigenicity and the immunogenicity of the synthetic glycoconjugates were investigated.

EXPERIMENTAL PROCEDURES * To whom correspondence should be addressed: Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201. Telephone: 410-706-4982. Fax: 410-706-5068. E-mail: [email protected].

General Methods. 1H NMR spectra were recorded on a 300 MHz machine with Me4Si (δ 0) as the internal standard. The ESI-MS spectra were measured on a Micromass ZQ-4000 single

10.1021/bc0502816 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/21/2006

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Figure 1. Schematic depiction of the designed glycoconjugate vaccine.

quadruple mass spectrometer. TLC was performed on glass plates coated with silica gel 60 F254 by visualizing the spots with UV light (254 nm) irradiation, 10% ethanolic sulfuric acid (for carbohydrates), ninhydrin spraying (for amines), and/or iodine coloration (for allyl groups). Flash column chromatography was performed on silica gel 60 (230-400 mesh). Photoaddition reaction was carried out in a quartz flask under N2. Analytical HPLC was carried out with a Waters 626 HPLC instrument on a Waters Nova-Pak C18 column (3.9 × 150 mm) at 40 °C. The column was eluted with a linear gradient of MeCN-water containing 0.1% TFA at a flow rate of 1 mL/ min with UV (214 nm) detection: Method A, 0-50% MeCN in 25 min; Method B, 0-90% MeCN in 25 min. Preparative HPLC was performed on a Waters 600 HPLC instrument equipped with a Waters C18 column (Symmetry 300, 19 × 300 mm). The column was eluted with a suitable gradient of MeCN-water containing 0.1% TFA at 12 mL/min. 2′-Azidoethyl 2,3,4,6-tetra-O-allyl-R-D-galactopyranoside (2). A solution of 2-azidoethyl R-D-galactopyranoside (1) (0.96 g, 3.85 mmol) in anhydrous DMF (5 mL) was added dropwise to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 1.23 g, 30.80 mmol) in anhydrous DMF (30 mL). After being stirred for 30 min at room temperature, the reaction mixture was cooled to 0 °C, and then allyl bromide (3.70 g, 30.80 mmol) was added dropwise. The resulting mixture was stirred for 1 h at 0 °C and for 3 h at room temperature. After the mixture was cooled with an ice bath, the excess sodium hydride was quenched by the slow addition of methanol (5 mL). The volatile was evaporated to dryness, and then the residue was mixed with ethyl acetate (200 mL) and washed with brine (3 × 50 mL). The organic phase was dried over Na2SO4, filtered, and concentrated at a reduced pressure. The residue was purified by flash chromatography (hexane/EtOAc 80:20) to give compound 2 (1.39 g, 88%). Rf 0.46 (hexane/EtOAc 80:20); 1H NMR (300 MHz, CDCl3/TMS): δ 6.05-5.84 (m, 4 H, OCH2CHd CH2), 5.36-5.14 (m, 8 H, OCH2CHdCH2), 4.95 (d, 1 H, J ) 3.2 Hz, H-1), 4.39 (dd, 1 H, J ) 12.6, 5.5 Hz, OCHHCHd CH2),), 4.26 (dd, 1 H, J ) 12.6, 5.2 Hz, OCHHCHdCH2), 4.22-4.06 (m, 4 H, OCH2CHdCH2), 4.04-3.98 (m, 2 H, OCH2CHdCH2), 3.96 (dd, 1 H, J ) 6.6, 6.4 Hz, H-5),), 4.013.80 (m, 3 H, H-2, H-4 and OCHHCH2N3), 3.75 (dd, 1 H, J ) 10.1, 2.8, H-3), 3.67 (ddd, 1 H, J ) 10.8, 5.9, 3.9 Hz, OCHHCH2N3), 3.62-3.51 (m, 3 H, H-6, H-6′, and OCH2CHHN3), 3.38 (ddd, 1 H, J ) 13.2, 5.6, 3.9 Hz, OCH2CHHN3); ESI-MS (m/z): 432.41 (M + Na)+. 2′-(6-tert-Boc-Aminohexoic-amido)-ethyl 2,3,4,6-Tetra-Oallyl-R-D-galactopyranoside (3). To a stirred mixture of azide

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2 (269.9 mg, 0.66 mmol) was added N-Boc-6-aminocaproic acid (191.6 mg, 0.83 mmol) in toluene (10 mL) n-Bu3P (167.6 mg, 0.83 mmol) dropwise. After being stirred at room temperature for 3 h and then at 100 °C for 18 h, the resulting mixture was cooled to room temperature, and the solvent was evaporated at reduced pressure. The residue was purified by flash column chromatography (CH2Cl2/MeOH 95:5) to give compound 3 (180.9 mg. 46%), Rf 0.44 (CH2Cl2/MeOH, 95:5); 1H NMR (CDCl3/TMS): δ 6.63 (t, 1 H, J ) 6.4 Hz, NH), 6.02-5.82 (m, 4 H, OCH2CHdCH2), 5.36-5.14 (m, 8 H, OCH2CHdCH2), 4.90 (d, 1 H, J ) 3.2 Hz, H-1), 4.60 (t, 1 H, J ) 6.2 Hz, NHBoct), 4.38 (dd, 1 H, J ) 11.7, 5.8 Hz, OCHHCHdCH2), 4.27 (dd, 1 H, J ) 11.7, 5.8 Hz, OCHHCHdCH2), 4.22-3.94 (m, 6 H, OCHHCHdCH2), 3.90-3.78 (m, 3 H, H-2, H-4, OCHHCH2NH), 3.76-3.66 (m, 3 H, H-3, H-5, OCHHCH2NH), 3.58 (d, 2 H, J ) 6.2 Hz, H-6, H-6′), 3.52-3.38 (m, 2 H, OCH2CH2NH), 3.14-3.06 (m, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 2.15 (t, J ) 7.1 Hz, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 1.721.55 (m, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 1.44 (s, 9 H, H-Boc-t), 1.43-1.30 (m, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 0.94 (p, J ) 6.7 Hz, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t); ESI-MS (m/z): 619.32 (M + Na)+, 597.27 (M + H)+, 497.30 (M - Boc + H)+. 2′-(6-tert-Boc-Aminohexoic-amido)-ethyl 2,3,4,6-Tetra-O(6-amino-3-thiahexyl)-R-D-galactopyranoside (4). To a solution of compound 3 (173.0 mg, 0.29 mmol) and AIBN (20.0 mg) in methanol (15 mL) in a Quartz flask was added cysteamine hydrochloride (263.6 g, 2.32 mmol). After being degassed by bubbling N2 into solution for 30 min, the resulting mixture was stirred and irradiated (UV, 254 nm) under N2 for 24 h. The volatile was evaporated under reduced pressure, and the residue was purified by gel filtration on Sephadex G-15 column (2.5 × 75 cm) using water as eluent. Fractions containing the product were pooled and lyophilized to give compound 4 (859.5 mg, 82%). 1H NMR (D2O): δ 5.09 (d, 1 H, J ) 3.4 Hz, H-1), 4.04-3.33 (m, 16 H, H-2, H-3, H-4, H-5, H-6, H-6′, OCH2CH2CH2S, and OCH2CH2NH), 3.26-3.14 (m, 10 H, SCH2CH2CH2NH2HCl, and OCH2CH2NH), 3.04 (t, 2 H, J ) 6.4 Hz, (CH2)4CH2NHBoc-t), 2.90-2.82 (m, 8 H, SCH2CH2CH2NH2HCl), 2.74-2.62 (m, 8 H, OCH2CH2CH2S), 2.25 (t, J ) 6.8 Hz, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 1.961.80 (m, 8 H, OCH2CH2CH2S), 1.66-1.55 (m, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 1.41 (s, 9 H, H-Boc-t), 1.38-1.26 (m, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t), 0.88 (p, J ) 6.2 Hz, 2 H, (O)CCH2CH2CH2CH2CH2NHBoc-t); ESIMS (m/z): 927.44 (M - 4HCl + Na)+, 905.53 (M - 4HCl + H)+, 453.51 (M - 4HCl + 2H)2+, 403.37 (M - 4HCl Boc + 2H)2+. 2′-(6-tert-Boc-Aminohexoic-amido)-ethyl 2,3,4,6-Tetra-O(6-(6-maleimido-hexanamido)-3-thiahexyl)-R-D-galactopyranoside (5). To a stirred solution of N-succimidyl 6-maleimidohexanoic acid ester (50.2 mg, 162.90 µmol) in THF (3 mL) at 0-5 °C was added a solution of 4 (21.4 mg, 20.36 µmol) in 0.1 M aqueous solution of NaHCO3 (1.0 mL). The resulting mixture was stirred for 1 h at 0-5 °C, then diluted with CHCl3 (50 mL) and washed with brine (3 × 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (CH2Cl2/MeOH 95:5) to give compound 5 (14.9 mg, 44%); Rf 0.57 (CH2Cl2/MeOH 90: 10); 1H NMR (CDCl3/TMS): δ 6.70 (s, 8 H, CHdCH), 6.62 (t, 1 H, J ) 6.4 Hz, NH), 6.36-6.16 (m, 4 H, NH), 4.89 (d, 1 H, J ) 3.0 Hz, H-1), 4.80-4.68 (t, 1 H, J ) 6.0 Hz, NHBoc-t), 3.94-3.84 (m, 2 H, OCH2CH2CH2), 3.80-3.34 (m, 32 H, H-2, H-3, H-4, H-5, H-6, H-6′, OCH2CH2CH2S, SCH2CH2NH, (CH2)4CH2N, and OCH2CH2NH), 3.18-3.08 (m, 2 H, (CH2)4CH2NHBoc-t), 2.74-2.55 (m, 16 H, OCH2CH2SCH2CH2NH), 2.28-2.14 (m, 10 H, (O)CCH2CH2CH2CH2CH2), 1.94-1.78

Carbohydrate-Based HIV-1 Vaccine

(m, 8 H, OCH2CH2CH2SCH2CH2N), 1.74-1.54 (m, 20 H, OCCH2CH2CH2CH2CH2N), 1.44 (s, 9 H, H-Boc-t), 1.40-1.24 (m, 10 H, (O)CCH2CH2CH2CH2CH2N, (O)CCH2CH2CH2CH2CH2NHBoc-t); ESI-MS (m/z): 1699.79 (M + Na)+, 1677.73 (M + H)+, 839.52 (M + 2H)2+, 789.55 (M - Boc + 2H)2+. The Oligomannose Cluster Carrying a Boc-Protected Amino Group (6). To a solution of the SH-tagged oligomannose Man9GlcNAc2Asn-SH (18.0 mg, 8.69 µmol) (27) in a phosphate buffer (pH 6.6, 50 mM, 3.0 mL) and acetonitrile (3.0 mL) was added a solution of the scaffold 5 (1.83 mg, 1.09 µmol) in DMF (104 µL). The resulting mixture was shaken gently at room-temperature overnight. The mixture was lyophilized to dryness, and the ligation product was purified by preparative reverse-phase HPLC to yield the tetravalent oligomannose derivative 6 (10.9 mg, 68%): analytical HPLC (Method A), tR ) 16.71 min; ESI-MS: 2492.39 (M + 4H)4+, 1994.05 (M + 5H)5+, 1974.11 (M - Boc + 5H)5+, 1961.41 (M Man + 5H)5+, 1941.74 (M - Boc-Man + 5H)5+, 1929.31 (M - 2Man + 5H)5+. Oligomannose Cluster Carrying a Free Amino Group (7). A solution of the Boc-containing oligomannose cluster 6 (10.0 mg, 1.00 µmol) in a mixture of TFA, acetonitrile, and water (10/10/1, v/v, 2 mL) was shaken at room temperature for 2 h. The mixture was lyophilized to dryness, and the residue was purified by preparative HPLC to give the oligomannose cluster 7 (8.9 mg, 90%): Analytical HPLC (Method A), tR ) 14.63 min; ESI-MS (m/z): 2467.33 (M + 4H)4+, 1973.97 (M + 5H)5+, 1941.80 (M - Man + 5H)5+, 1909.30 (M - 2Man + 5H)5+. Maleimido-Functionalized Oligomannose Cluster (8). Compound 7 (8.5 mg, 0.86 µmol) was dissolved in a mixed solvent of a phosphate buffer (pH 7.4, 50 mM) and acetonitrile (2/1, v/v, 1.5 mL). To the solution was added N-succimidyl 6-maleimidohexanoic ester (2.7 mg, 8.60 µmol) in acetonitrile (0.5 mL). The reaction mixture was stirred at room temperature for 2 h and then lyophilized to dryness. The residue was purified by preparative HPLC to yield the desired maleimido-functionalized oligomannose cluster (8) (5.9 mg, 68%): Analytical HPLC (Method A), tR ) 16.36 min; ESI-MS (m/z): 2516.45 (M + 4H)4+, 2013.27 (M + 5H)5+, 1980.75 (M - Man + 5H)5+, 1948.22 (M - 2Man + 5H)5+. Biotinylated Oligomannose Cluster (9). Compound 7 (2.2 mg, 0.22 µmol) was dissolved in a mixed solvent of a phosphate buffer (pH 7.4, 50 mM) and acetonitrile (2/1, v/v, 1.0 mL). Then NHS-LS-biotin (5 equiv) was added, and the mixture was stirred at room temperature for 2 h. The mixture was lyophilized, and the residue was purified by preparative HPLC to give the M9Cluster-X-biotin (9) (1.88 mg, 84%): Analytical HPLC (Method A), tR ) 15.81 min; ESI-MS: 2552.52 (M + 4H)4+, 2041.77 (M + 5H)5+, 2009.205 (M - Man + 5H)5+, 1977.50 (M - 2Man + 5H)5+. Oligomannose Cluster/T-Helper Peptide Conjugate (10). A mixture of the maleimido-M9Cluster (8) (2 mg, 0.2 µmol) and the cysteine-containing tetanus toxoid peptide (TT-peptide) CGQYIKANSKFIGITEL (32) (0.76 mg, 0.4 µmol) in a mixed solvent containing phosphate buffer (pH 6.6, 50 mM) and acetonitrile (1/1, 1 mL) was kept at room temperature for 2 h with gentle shaking. The reaction mixture was lyophilized to dryness. The residue was subject to preparative RP-HPLC to give the glycopeptide M9Cluster-Pep (10) (1.9 mg, 79%): Analytical HPLC (Method B), tR ) 11.49 min; ESI-MS: 2389.39 (M + 5H)5+, 2357.21 (M - Man + 5H)5+, 1991.38 (M + 6H)6+, 1964.55 (M - Man + 6H)6+, 1707.08 (M + 7H)7+, 1684.01 (M - Man + 7H)7+. Oligomannose Cluster/KLH Conjugate (11). A mixture of thiolated KLH (10 mg), prepared according to the reported procedure (33), and the maleimido-M9Cluster 8 (2.2 mg) in a mixed solvent of phosphate buffer (pH 6.6, 50 mM) and

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acetonitrile (1/1, 3 mL) containing EDTA (5 mM) was incubated at room temperature for 4 h with gentle shaking. Then a solution of cysteine (0.1 M, 100 µL) in a phosphate buffer was added to quench any residual maleimide on KLH. The solution was lyophilized, and the conjugate M9Cluster-KLH (11) was isolated by gel filtration on a Sephadex G-15 column equilibrated and eluted with a phosphate buffer (10 mM, pH 7.2). The carbohydrate content was measured by anthrone assay, and the protein content was measured using BioRad dye reagent according to the manufacturer’s instructions. Man9/T-Helper Peptide Conjugate (13). A mixture of the maleimido-Man9 (34) (2.5 mg, 1.14 µmol) and the cysteinecontaining tetanus toxoid peptide (TT-peptide) (5.4 mg, 2.8 µmol) in a mixed solvent containing phosphate buffer (pH 6.6, 50 mM) and acetonitrile (1/1, 1 mL) was kept at room temperature for 2 h with gentle shaking. The reaction mixture was lyophilized and the residue was subject to preparative RPHPLC to give the glycopeptide Man9-Pep (13) (3.7 mg, 81%): Analytical HPLC (Method B), tR ) 11.37 min; ESI-MS: 1359.27 (M + 3H)3+, 1305.27 (M - Man+3H)3+, 1251.27 (M - 2Man+3H)3+, 1019.73 (M + 4H)4+, 979.24 (M Man+4H)4+, 938.62 (M - 2Man+4H)4+. Man9/KLH Conjugate (14). A mixture of thiolated KLH (5 mg) and the maleimido-Man9 (1.5 mg) in a mixed solvent of phosphate buffer (pH 6.6, 50 mM) and acetonitrile (1/1, 2 mL) containing EDTA (5 mM) was incubated at room temperature for 4 h with gentle shaking. Then a solution of cysteine (0.1 M, 50 µL) in a phosphate buffer was added to quench any residual maleimide on KLH. The solution was lyophilized, and the conjugate Man9-KLH (14) was isolated by gel filtration on a Sephadex G-15 column equilibrated and eluted with a phosphate buffer (10 mM, pH 7.2). The carbohydrate content was measured by anthrone assay and the protein content was measured using BioRad dye reagent according to the manufacturer’s instructions. Biotinylated Man9 (15). A mixture of Man9GlcNAc2Asn (3 mg, 1.5 µmol) and NHS-LS-biotin (4.5 µmol) in a mixed solvent of phosphate buffer (pH 7.4, 50 mM) and acetonitrile (2/1, v/v, 1.0 mL) was stirred at room temperature for 3 h. The mixture was lyophilized and the residue was purified by preparative HPLC to give the Man9-X-biotin (15) (2.17 mg, 62%): Analytical HPLC (Method A), tR ) 8.97 min; ESI-MS: 2337.60 (M + H)+, 1169.16 (M + 2H)2+, 1088.40 (M Man + 2H)2+, 1007.12 (M - 2Man + 2H)2+, 926.09 (M 3Man + 2H)2+, 845.12 (M - 4Man + 2H)2+, 764.02 (M 5Man + 2H)2+. Rabbit Immunizations. New Zealand white rabbits (two rabbits per group) were used for the immunization studies. One milligram of respective glycoconjugate in 500 µL of water was emulsified with 500 µL of complete Freunds’ adjuvant (CFA). The emulsions were used for immunizing rabbits (two rabbits per group) subcutaneously. The rabbits were then boosted three times at days 14, 28, and 56, with each rabbit receiving at the intervals 0.5 mg of each glycoconjugate in 500 µL water emulsified with 500 µl of incomplete Freunds’ adjuvant (IFA). The rabbits were bled 10 days after the last boosting and the sera were obtained for the assays. Virus Neutralization Assay. The neutralizing activity of the sera was examined by the previously described method (35). Briefly, HIV-1IIIB (100 TCID50) was preincubated with serial dilutions of the rabbit anti-sera in RPMI 1640 containing 15% FCS for 45 min at 37 °C. Then 1.5 × 105 PHA-activated PBMCs were added, and the mixture were incubated for 24 h at 37 °C. The infected cells were washed five times with the complete medium to remove unabsorbed virus and residual antibody. Cells were resuspended in the complete medium containing IL-2 and were distributed into wells of a 96-well

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Scheme 1. Synthesis of Maleimide- and Biotin-tagged Oligomannose Clustersa

a Reaction conditions: (i) allyl bromide, NaH, DMF, 88%; (ii) N-Boc-6-aminocaproic acid, Bu P, toluene, 46%; (iii) cysteamine hydrochloride, 3 AIBN, MeOH, UV (254 nm), 82%; (iv) 6-maleimidohexanoic acid N-hydroxysuccinimide ester, THF, 0.1 M NaHCO3, 44%; (v) phosphate buffer (pH 6.6, 50 mM)/MeCN (1:1, v/v), 68%; (vi) TFA/MeCN/H2O (10:10:1, v/v), 90%; (vii) 6-maleimidohexanoic acid N-hydroxylsuccimide ester, phosphate buffer (pH 7.4, 50 mM)/MeCN (1:1, v/v), 68%; (viii) NHS-LC-biotin, phosphate buffer (pH 7.4)/MeCN, 84%.

tissue plate. Aliquots were taken from the culture on day 4 and 6. Viral growth was determined by measuring HIV-1 p24 antigen released into the culture medium by ELISA. The extent of neutralization activity was determined by measuring and comparing the p24 antigens released from the cells that were infected by the virus in the presence and absence of the rabbit sera or antibody. Human antibody 2G12 was used as a positive control for the neutralizing activity. Enzyme-Linked Immunosorbent Assay (ELISA). For biotinylated antigens (Man9-X-biotin, M9Cluster-x-biotin, G-Xbiotin, and GCluster-X-biotin), the 96-well ELISA microtiter plates were first coated with 8 µg/mL Neutravidin (streptavidin) in PBS and incubated at 4°C overnight. After washings with PBS/0.5% Tween-20, nonspecific binding was blocked with 5% (w/v) BSA in PBS at room temperature for 1 h. Plates were washed, and then 5 µg/mL of the respective biotinylated antigen in 1% BSA was added. Plates were incubated at 37 °C for 1 h. The plates were again washed, and rabbit sera were added in a 1:2 or 1:3 serial dilution in 1% BSA/PBS. The plates were incubated at 37°C for 2 h and washed. To the plates was added a solution (100 µL) of 1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H & L) in 1% BSA/ PBS. After incubation for 1 h at 37 °C, the plates were washed again and a solution of 3,3′,5,5′-tetramethylbenzidine (TMB) was added. Color was allowed to develop for 5 min, and the reaction was then quenched by adding a solution of 0.5 M H2SO4 to each well. The optical density was then measured at 450 nm. For testing the cross-reactivity of the sera to HIV-1

gp120, 10 µg/mL of HIV-1 (BAL) gp120 (produced in human H9 cell lines) was coated to the 96-well plates, and antibody titers were measured as described above. For testing the binding of the synthetic glycoconjugates with human antibody 2G12, the 96-well microtiter plates were incubated with 10 µg/mL of Man9-KLH and M9Cluster-KLH, and then the plates were titrated against a serial dilution of human antibody 2G12 (obtained from the NIH AIDS Research and Reference Reagent Program) starting at 10 µg/mL. After washings, the plates were incubated with a solution (100 µL) of 1:3000 diluted horseradish peroxidase (HRP)-conjugated goat anti-human IgG in 1% BSA/ PBS. Color was developed and the OD450 was read.

RESULTS AND DISCUSSION Synthesis. The construction of the glycoconjugate vaccines requires chemoselective conjugation of the galactose-based oligomannose cluster (Tetra-Man9) to a carrier protein or to a T-helper epitope peptide. For this purpose, the galactose scaffold was modified at the aglycon portion so that a suitable functional group could be selectively generated at later stage for ligation without influencing the integrity of the assembled oligomannose cluster. The synthesis started with the 2-azidoethyl R-Dgalactoside (1), which was prepared by reaction of D-galactose with 2-chloroethanallyl alcohol under the catalysis of BF3‚Et2O with subsequent displacement of the chloride with azide (34). Treatment of 1 with sodium hydride and allyl bromide in N,Ndimethylformamide gave the O-allylated 2-azidoethyl galacto-

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Carbohydrate-Based HIV-1 Vaccine Scheme 2. Synthesis of Oligomannose Cluster-Peptide/Protein Conjugates

side 2. Reaction of 2 with N-Boc-6-aminocarproic acid in the presence of tributylphosphine gave the derivative 3. Introduction of four maleimido groups was achieved via a two-step conversion: the photoaddition of cysteamines to the allyl functional groups in 3 to give compound 4, and subsequent elongation of the spacer by reaction of the free amino groups with the active ester of 6-maleimidohexanoic acid to provide the maleimido derivative 5 (Scheme 1). The ligation of the maleimidofunctionalized scaffold with the SH-tagged Man9GlcNAc2Asn, which was prepared according to the literature (27), was performed in phosphate buffer (pH 6.6) containing MeCN to give the oligomannose cluster 6 in 68% yield, which was purified by HPLC and characterized by ESI-MS. The N-Boc group was selectively removed by treatment with TFAMeCN-H2O (10:10:1, v/v) to yield compound 7 that carries a free amino group. Treatment of 7 with 6-maleimidohexanoic acid N-hydroxylsuccinimide ester gave the maleimido-functionalized oligomannose cluster 8 in 68% yield. Selective biotinylation of 7 with active ester NHS-LC-biotin afforded the biotin-tagged oligomannose cluster (M9Cluster-X-biotin) (9) in 84% yield, which would be used as the coating antigen (Scheme 1). The synthesis of the oligomannose cluster-containing glycoconjugates was achieved through the chemoselective ligation

of the maleimido-functionalized oligomannose cluster (8) with the respective thiol-species under a mild aqueous condition. Briefly, chemoselective ligation between maleimide 8 and the T-helper peptide CGQYIKANSKFIGITEL from tetanus toxoid (aa830-844) (32) in a phosphate buffer (pH 6.6) containing MeCN gave the glycopeptide conjugate 10 (M9Cluster-Pep) in 79% yield, which was purified by HPLC and characterized by ESI-MS. On the other hand, treatment of 8 with thiolated KLH (33) in a phosphate buffer gave the KLH conjugate 11, M9Cluster-KLH, which was isolated by gel filtration (Scheme 2). The corresponding Man9-containing glycopeptide conjugate 13, Man9-Pep, was synthesized by chemoselective ligation between maleimido-functionalized Man9 (12) (34) and the TTpeptide (81% yield). Reaction of the maleimide-Man9 (12) with the thiolated KLH afforded the Man9-KLH conjugate 14 (Scheme 3). The carbohydrate contents of the KLH conjugates were determined by carbohydrate and protein assays. The synthetic M9Cluster-KLH (11) was determined to contain 15% carbohydrate, whereas the Man9-KLH (13) contained 19% carbohydrate. Finally, the coating antigen, Man9-X-biotin (15), was synthesized by treatment of Man9GlcNAc2Asn with NHSLC-biotin (Scheme 3). Binding of the Synthetic Glycoconjugates to Antibody 2G12. The 2G12 binding ability of the synthetic glycoconjugates

498 Bioconjugate Chem., Vol. 17, No. 2, 2006

Ni et al.

Scheme 3. Synthesis of Man9-KLH, Man9-Peptide, and Man9-Biotin Conjugatesa

a Reaction conditions: (i) Cys-tagged T-helper peptide, phosphate buffer (pH 6.6)/MeCN (1:1, v/v), 81%; (ii) thiolated KLH, phosphate buffer (pH 7.2), EDTA; (iii) NHS-LC-biotin (Pierce), phosphate buffer (pH 7.4, 50 mM)/MeCN (1:1, v/v), 62%.

antigens than the monomeric Man9 oligomannose (27, 28, 30). Interestingly, the synthetic Man9-KLH conjugate, which contains 19% Man9 (by weight), was able to bind to 2G12 (EC50 at ca. 6 µg/mL) (Figure 2B), demonstrating certain clustering effect of the Man9 moieties within the conjugate. The 2G12binding ability of M9Cluster-KLH is about the same as that of the M9Cluster-X-biotin under the same binding conditions (Figure 2, panels A and B). The results suggest that the mild chemoselective conjugation between the M9Cluster-maleimide and the thiolated KLH did not affect the structural integrity of the oligomannose cluster. Immunization Studies. We next evaluated the immunogenicity of the synthetic glycoconjugates in rabbits. Pre-sera were taken before the immunization and the anti-sera were collected after four immunizations (priming and three boosts). The sera were first tested against the respective antigen-X-biotin as the coating antigen. The ELISA results were summarized in Table 1. It was found that the KLH conjugates raised higher IgG type antibody titers than the corresponding peptide conjugates. Particularly, the M9Cluster-KLH raised unusually high antibody titer (>1:100 000). To examine the possibility that the antibodies might be raised against the linkers rather than the carbohydrate antigens, we prepared the corresponding biotin-tagged linkers (linker-X-biotin) by treatment of the biotin-tagged glycoconjugates with endo-β-N-acetyl-glucosaminidase from Arthrobacter (Endo-A). Endo-A is known to be able to remove the high-mannose type N-glycan (here, Man9GlcNAc) from gly-

Figure 2. Binding of antibody 2G12 to synthetic glycoconjugates. A, binding to biotinylated Man9 and Man9 cluster; B, binding to KLHconjugated Man9 and Man9 cluster.

was estimated by ELISAs. For the binding assays, the biotinylated coating antigens, M9Cluster-X-biotin (9) and Man9-Xbiotin (15), were immobilized on streptavidin plates, whereas the KLH conjugates (11) and (14) were coated directly to the plastic microtiter plates. The plates were then titrated with serial dilutions of antibody 2G12 (Figure 2). As shown in Figure 2A, the Man9-cluster was readily recognized by antibody 2G12 at an apparent EC50 of ca. 0.8 µg/mL under the assay condition. However, the immobilized monomeric Man9 was unable to catch 2G12 in the solution when the concentration of 2G12 was below 10 µg/mL. The results are consistent with the previous observations that synthetic Man9-clusters are much better Table 1. ELISA Antibody Titers against the Respective Coating Antigensa

synthetic immunogens

a

coating antigens

Man9-KLH

M9Cluster-KLH

Man9-Pep

M9Cluster-Pep

Man9-X-biotin G-X-biotin M9Cluster-X-biotin G-Cluster-X-biotin

960 460 n.d.b n.d.b

n.d.b n.d.b 163,840 81,920

640 480 n.d.b n.d.b

n.d.b n.d.b 2560 1280

The antibody titer was defined as the highest dilutions of the sera that yielded an absorbance (OD450) of 0.2 in the ELISA. b Not determined.

Bioconjugate Chem., Vol. 17, No. 2, 2006 499

Carbohydrate-Based HIV-1 Vaccine Table 2. ELISA Antibody Titers against HIV-1 gp120a synthetic immunogens rabbit sera Man9-KLH M9Cluster-KLH Man9-Pep M9Cluster-Pep pre-serab anti-sera

80 120

60 150

60 80

80 120

a The antibody titer was defined as the highest dilutions of the sera yielded an absorbance (OD450) of 0.2 in the ELISA. b The pre-sera were the sera taken from each of the rabbit group before immunizations.

coconjugates by cleaving the N-glycans at the GlcNAcβ1,4GlcNAc linkage (36, 37). As monitored by HPLC coupled with ESI-MS analysis, we found that incubation of the Man9-X-biotin and the Man9Cluster-X-biotin with Endo-A could completely remove the Man9 portions from the conjugates, giving the corresponding GlcNAc-X-biotin (G-X-biotin) and GlcNAcCluster-X-biotin (G-cluster-X-biotin), respectively. When the G-Xbiotin and G-Cluster-X-biotin were used as the coating antigens on a streptavidin plate, the ELISA assay revealed that the majority of the antibodies generated were in fact directed to the linkers, although a small fraction of the antibodies might be raised against the carbohydrate antigen (Table 1). The results suggest that the oligomannose (Man9) and the corresponding clustered oligomannose antigens were poorly immunogenic even when conjugated to KLH (a strong immune-stimulating carrier protein) or to the TT-peptide (a universal T-helper epitope). On the other hand, the fact that the majority of antibody responses were directed to the linkers strongly suggests that the choice of an appropriate linker in making the glycoconjugate vaccines is of primary importance for raising sufficient antibody responses against the weakly immunogenic carbohydrate antigen. Recently, an excellent study has also demonstrated that a maleimidefunctionalized linker significantly suppressed the immunogenicity of a tumor-associated carbohydrate antigen in its KLH conjugate (38), resulting in low antibody titers specific for the carbohydrate antigen. We also tested reactivity of the rabbit sera toward HIV-1 envelope glycoprotein gp120 (BAL gp120 expressed in human H9 cell lines) (Table 2). It was found that the rabbit pre-sera showed a significant background of cross-reactivity to the HIV-1 gp120. Nevertheless, the anti-sera clearly showed an enhanced reactivity to HIV-1 gp120 above the background of the presera (Table 2), which might be contributed from the oligomannose-specific antibodies in the anti-sera. A preliminary HIVneutralization assay indicated that, at 1:10 dilution, none of the rabbit anti-sera demonstrated HIV-1 neutralizing activities. The results implicated that either the glycoconjugates failed to raise any neutralizing antibodies under the immunization protocol or, if any, the neutralizing antibodies might be generated only at very low levels insufficient to show neutralizing activities.

CONCLUSION The recent characterization of the neutralizing epitope of the broadly neutralizing antibody 2G12 as a novel oligomannose cluster on the envelope glycoprotein gp120 has implicated the possibility of developing a carbohydrate-based HIV-1 vaccine. The present work represents the first attempt to create such a vaccine by synthesizing oligomannose- and oligomannose cluster-containing glycoconjugates. The synthetic oligomannose cluster could be recognized by the human antibody 2G12 in binding studies. Preliminary immunization studies suggested that moderate carbohydrate-specific antibodies were raised by the glycoconjugate immunogens, but the majority of antibody responses were directed to the linkers. In addition, the antisera were weakly cross-reactive to HIV-1 gp120, but the carbohydrate-specific antibodies in the anti-sera generated by our current immunization protocol have not reached the level that could neutralize HIV-1 infection. The results implicated

the poor immunogenicity of the carbohydrate antigens and, again, attested to the difficulties of raising neutralizing antibodies against HIV-1 in general. It is suggested that the next step should focus on enhancing the immunogenicity of the HIV-1 carbohydrate antigen, such as synthesizing more effective glycoconjugate immunogen (better linkers and more effective built-in adjuvant), optimizing the immunization protocols, and emphasizing fundamental studies on B cell immunology to break the immune tolerance on the HIV-1 carbohydrate antigens.

ACKNOWLEDGMENT We thank Prof. Anthony L. DeVico, Prof. George K. Lewis, Dr. Shibo Jiang, and Dr. Robert Powell for helpful discussions during the work. The human antibody 2G12 was obtained from the National Institutes of Health (NIH)’s AIDS Research and Reference Reagent Program. The work was supported by the National Institutes of Health (NIH grant R21 AI054354). Supporting Information Available: The HPLC profiles and ESI-MS spectra of the synthetic, homogeneous glycopeptides. This material is available free of charge via the Internet at http:// pubs.acs.org/BC.

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