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Journal of Virology, July 2008, p. 6359-6368, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00293-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120

Rena D. Astronomo,¹ Hing-Ken Lee,^3,4, Christopher N. Scanlan,^1,6 Ralph Pantophlet,¹ Cheng-Yuan Huang,^3,4, Ian A. Wilson,^2,3 Ola Blixt,^2,5, Raymond A. Dwek,⁶ Chi-Huey Wong,^3,4 and Dennis R. Burton^1,2*

Department of Immunology and Microbial Science,¹ Department of Molecular Biology,² The Skaggs Institute for Chemical Biology,³ Department of Chemistry,⁴ the Glycan Array Synthesis Core D, Consortium for Functional Glycomics, The Scripps Research Institute, La Jolla, California 92037,⁵ The Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom⁶

Received 8 February 2008/ Accepted 14 April 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man₄) that corresponds to the D1 arm of Man₉GlcNAc₂ inhibits 2G12 binding to gp120 as efficiently as Man₉GlcNAc₂ itself, indicating the potential use of Man₄ as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man₄ on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man₄ up to a limit which is achieved at 10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man₄)₁₄ elicits significant serum Ab titers to Man₄. However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man₉ derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man₄ on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Approximately 30 million people are infected with human immunodeficiency virus (HIV), and new infections continue at a high rate (75). An effective vaccine is urgently needed to curb this pandemic. The prevailing view is that such a vaccine should elicit both neutralizing antibodies (NAbs) and T-cell responses against HIV (32, 38, 45). Virtually all vaccine initiatives to elicit NAbs are focused on the external envelope glycoproteins, gp120 and gp41 (10, 50). The natural immune response to HIV is generally characterized by titers of NAbs against a narrow range of isolates (54, 70); broadly NAbs arise only in some individuals over time (3, 12, 21, 40). To date, immunogens capable of eliciting broadly NAb responses have not been forthcoming despite extensive efforts (12, 79). However, a small panel of broadly NAbs has been isolated from HIV-1-infected individuals (2, 4, 11-13, 18, 19, 47, 65, 82) and shown to provide protection against viral challenge in animal models (1, 25, 27, 30, 42, 43, 49, 51, 52, 61). The epitopes of these Abs may serve as targets for vaccine design (9, 14, 22).

The difficulties in eliciting broadly NAbs may be largely attributed to the nature of the neutralization target on HIV-1—a compact, heavily glycosylated envelope trimer wherein conserved surfaces are either recessed or otherwise difficult for Abs to access (17, 35, 36, 50, 55, 59, 76-78, 80). The main solvent-accessible face of gp120 is decorated by a dense array of carbohydrates, the so-called "glycan shield," that masks conserved protein epitopes (6, 44, 73). This face of gp120 (the "silent face") is expected to be poorly immunogenic for a number of reasons. First, glycoproteins exist as numerous glycoforms in which different glycans can exist at a single site, thereby providing a heterogeneous array of antigens and diluting out the antiglycan immune response to any single glycoform (56). Second, the individual glycans attached to viral proteins by the host glycosylation machinery are identical to glycans found on self proteins and are expected to be subject to tolerance mechanisms. Third, protein-glycan interactions are typically weaker than interactions among proteins, potentially restricting the level of affinity maturation available to antiglycan Abs (62, 71, 72).

Nevertheless, one of the HIV-1 broadly NAbs, 2G12, recognizes a conserved carbohydrate epitope corresponding to a unique cluster of oligomannose residues associated with the glycan shield of gp120 (8, 34, 57, 60, 65). Such a dense array of high-mannose glycans has not been observed among human glycoproteins. Moreover, 2G12 neutralizes a broad spectrum of HIV-1 isolates across different clades in vitro (4, 23, 64, 65), protects against infection in monkey models, and exerts selection pressure on virus in humans while being well tolerated (43, 46, 63). Such studies suggest that targeting the conserved oligomannose clusters on gp120 is a potential vaccine strategy. Toward this goal, the fine specificity of 2G12 has been characterized structurally and biochemically with chemically defined oligomannoses and/or related synthetic oligosaccharide clusters by a number of groups (7, 15, 16, 24, 28, 37, 41, 53, 69). Crystal structures of Fab 2G12 alone and in complex with various oligomannosides, including Man₉GlcNAc₂ and synthetic glycans, revealed the unconventional architecture of 2G12 in which two Fab fragments interlock via heavy chain variable region (V_H) domain swapping, creating a novel V_H-V_H interface in addition to the two conventional V_H-V_L (where L indicates the light chain) binding sites (15, 16). The conventional antigen-combining sites interact specifically with the terminal Man12Man moieties on the D1 or D3 arms of high-mannose glycans (15, 16).

The elucidation of the 2G12 carbohydrate epitope engendered a keen interest in the design of immunogens to target the glycan shield using 2G12 as a template. While carbohydrates normally fail to induce long-lived, Ab-mediated protection, conjugation of glycans to protein scaffolds has generated successful vaccines capable of inducing protection against a number of bacterial infections (66). Several synthetic mannose antigens have been created as 2G12 epitope mimics for the development of a carbohydrate-based HIV-1 vaccine component (33, 39, 48, 67, 69). Multivalent presentations of Man₉GlcNAc₂ on various scaffolds and, more recently, D1 arm tetramannosides on a regioselectively addressable functionalized template have been synthesized and shown to be more effective in binding to 2G12 than monomeric Man₉GlcNAc₂ (or Man₉GlcNAc₂Asn) (67). However, no glycoconjugate construct has yet successfully elicited reasonable titers against the displayed glycans that also cross-react with gp120. Herein, the development of multivalent, neoglycoconjugate antigens in which a synthetic D1 arm tetramannoside is directly conjugated to bovine serum albumin (BSA) is described. The antigenicity and immunogenicity of a multivalently displayed Man₄ derivative of Man₉GlcNAc₂ are investigated.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
General procedures for conjugation of Man₄ to carrier proteins. The synthesis of the Man₄ building block used here has been previously described (37). NaHCO₃ (3.5 mg) and Na₂CO₃ (2.5 mg) were added to an aqueous Man₄ solution (5 mg of Man₄ in 0.2 ml of H₂O) undergoing constant stirring (Fig. 1). The solution was adjusted to pH 9 by adding solid Na₂CO₃. Then, 10 µl of thiophosgene was dissolved in CHCl₃ (0.5 ml), and this thiophosgene solution (0.15 ml in CHCl₃) was injected into the mannose solution all at once. The solution mixture was stirred vigorously at room temperature for 2.5 h. Completion of the reaction was determined by a negative ninhydrin test. The organic phase was removed under reduced pressure (in a fume hood) from the aqueous solution, which was used without further purification.

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FIG. 1. Synthetic scheme for the conjugation of Man4 to BSA. n, number of Man4 moieties conjugated to different Lys residues on BSA.

BSA (2 mg) was dissolved in 0.3 ml of 0.3 M NaHCO₃ (buffer) followed by the addition of the above activated sugar solution in one portion, approximately 20 times the activated sugars per Lys residue on BSA to generate the species BSA-(Man₄)₁₄. Other BSA-(Man₄)_n species (where n is the number of Man₄ moieties conjugated to different Lys residues on BSA) were generated using different ratios (5-, 10-, and 15-fold molar excess) of activated sugar per Lys residue on BSA (Table 1), while the ovalbumin (Ova) glycoconjugate, Ova-(Man₄)₁₀, was prepared in the same manner as BSA-(Man₄)₁₄. The alkalinity of the solution was adjusted to pH 9 by adding solid Na₂CO₃. The reaction was allowed to proceed with constant stirring for 12 h, after which the molecular weight of the conjugated protein was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The protein was then purified by Econo-Pac 10DG columns and eluted with phosphate-buffered saline (PBS), with detection performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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TABLE 1. Relationship between copy number of Man4 on BSA glycoconjugates and 2G12 binding as measured by the ability of the glycoconjugates to inhibit 2G12 binding to gp120

Immunization of rabbits with BSA-(Man₄)₁₄. New Zealand White rabbits received either BSA-(Man₄)₁₄ or BSA mixed 1:1 with Ribi adjuvant (R-730 [monophosphoryl lipid A, synthetic trehalose dicorynomycolate, cell wall skeleton]; Corixa) such that each rabbit (four per group) per immunization received 200 µg (total volume of 1 ml) of glycoconjugate or carrier protein alone via subcutaneous and intramuscular injection. After priming, monthly booster injections were given [three and four for the BSA and BSA-(Man₄)₁₄ groups, respectively], and bleeds were taken approximately 7 to 10 days postimmunization. Animal housing and immunization procedures adhered to the protocols of the Institutional Animal Care and Use Committee.

ELISA. Enzyme-linked immunosorbent assays (ELISAs) were performed to measure the binding of 2G12 and rabbit serum Abs to a number of antigens with and without soluble inhibitors. In all assays, development was monitored at 405 nm.

(i) 2G12 inhibition ELISA. Determination of 2G12 binding inhibition by the various BSA-glycoconjugates, gp120, and soluble mannosides was performed as previously described (60). Briefly, 50 ng of gp120 from HIV-1 strain JR-CSF ([gp120_JR-CSF] produced in-house from the CHO.FP.JR.F13 cell line [Maxygen] and purified on a Ni-nitrilotriacetic acid Superflow [Qiagen] column) was coated onto flat-bottomed microtiter plates (Costar type 3690; Corning Inc.) at 4°C overnight. Subsequent steps were done at room temperature. The plates were washed twice with PBS-0.05% Tween 20 (PBS-T) and then blocked with 3% BSA (100 µl/well) for 1 h. The wells were then emptied, and 2G12 (provided by Gabriela Stiegler and Hermann Katinger), diluted to 0.5 µg/ml with 1% BSA-0.02% Tween 20-PBS (PBS-BT), was added in the presence of molar titrations of the aforementioned inhibitors and incubated for 1 h. After the wells were washed, Ab binding was probed with alkaline phosphatase-conjugated goat anti-human immunoglobulin G (IgG) F(ab')₂ Ab (Pierce) diluted 1:1,000 in PBS-BT (1 h). The wells were again washed, and bound Ab was visualized with p-nitrophenol phosphate substrate (Sigma).

(ii) Serum ELISAs. Serum binding to gp120_JR-CSF, Ova, BSA-(Man₄)₁₄, and Ova-(Man₄)₁₀ was measured by ELISA using a similar, previously described protocol (15). Microtiter plate wells were coated with 250 ng of antigen overnight at 4°C. The wells were then washed and blocked as described above. Serial dilutions (starting at 1:100 in PBS-BT) of heat-inactivated (30 min at 56°C) serum were allowed to bind antigen for 1 h prior to washing. For determining the inhibition of BSA-(Man₄)₁₄ serum binding to cognate immunogen by soluble oligomannosides, serum from the final animal bleeds was used. Diluted serum (1:400) was allowed to bind BSA-(Man₄)₁₄ in the presence of serially diluted D-mannose (Sigma) and the synthetic oligomannosides Man₃, Man₄, and Man₇ for 2 h prior to washing. Ab binding was detected and visualized as described above, using alkaline phosphatase-conjugated goat anti-rabbit IgG F(ab')₂ Ab (Pierce). Absorbance was read 30 min after substrate addition.

Glycan recognition profiling of rabbit serum by glycan microarray analysis. Serum from BSA and BSA-(Man₄)₁₄-immunized rabbits was screened on a printed glycan array, version 3.0, from the Consortium for Functional Glycomics (CFG) according to standard protocols (5). Briefly, glycan array slides were rehydrated in PBS, followed by incubation with 1 ml of 1:200-diluted individual heat-inactivated serum samples (1 h) in a humidified chamber with gentle rocking. All incubations are performed likewise with 1-ml volumes prepared in PBS-T supplemented with 3% BSA. Samples were removed, and slides were washed by four successive rinses in PBS-T, PBS, and distilled H₂O. Serum IgG was detected using biotin-SP-conjugated goat anti-rabbit IgG Fc fragment-specific secondary antibody (6 µg/ml; Jackson ImmunoResearch Laboratories Inc.), and after another series of washes, bound Ab was visualized using streptavidin-Alexa 488 (0.04 µg/ml; Invitrogen). The slides were washed a final time (four rinses each in PBS-T and PBS and then three successive rinses in separate reservoirs of distilled H₂O) and then dried by low-speed centrifugation prior to image acquisition. Fluorescence intensity was measured using a ProScanArray HT Microarray Scanner (Perkin Elmer) confocal scanner, and image analyses were performed using Imagene image analysis software (BioDiscovery, El Segundo, CA) as described previously. The average over four replicates and the standard error of the mean for selected glycans were plotted using GraphPad Prism software. The highest and lowest data points out of six replicates printed on the chips were excluded to control for variation in the glycan printing process.

Complete glycan array data sets may be found at www.functionalglycomics.org in the CFG data archive under cfg_rRequest_517.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Multivalent presentation of Man₄ on BSA greatly enhances 2G12 affinity. The synthetic tetramannoside Man₄ (Man12Man12Man13Man) was previously shown to bind in the primary Ab-combining site of 2G12 and to be at least as effective an inhibitor of 2G12 binding as Man₉GlcNAc₂, a known ligand for 2G12 and possible constituent of the oligomannose cluster on gp120 recognized by 2G12 (15, 37). Thus, Man₄ is an attractive building block for the design of glycoconjugate immunogens for eliciting 2G12-like Abs. Herein, we describe the design of BSA neoglycoconjugates that multivalently display Man₄.

The relationship between the copy number of Man₄ molecules displayed on BSA and 2G12 binding was investigated using a panel of neoglycoconjugates that were synthesized at 5-, 10-, 15-, and 20-fold molar excesses of Man₄ to BSA to yield between 5 and 14 copies of Man₄ per BSA molecule. Increasing the average copy number of the displayed Man₄ molecules per BSA molecule improved 2G12 binding up to a threshold level, which was achieved at approximately 10 copies of Man₄ per BSA molecule (Table 1). At this valency, which is well below the theoretical loading capacity of 30 to 35 ligands per BSA molecule, the avidity effect appears to be maximized for 2G12 (29). The construct with the highest display number (n = 14) was termed BSA-(Man₄)₁₄ and was evaluated in parallel with D-mannose, soluble Man₄, and gp120 for its ability to inhibit 2G12 binding to its cognate epitope on gp120. The molar 50% inhibitory concentration (IC₅₀) values, as well as the molar IC₅₀ values normalized for the number of relevant mannose residues per ligand, are listed in Table 2. As shown previously (37), Man₄ is approximately 100-fold more potent as an inhibitor of 2G12 binding to gp120 than D-mannose. Notably, multivalent display of Man₄ on BSA resulted in an approximately 1,000-fold increase in 2G12 binding over the free glycan. However, its IC₅₀ is in the micromolar range, whereas 2G12 binds to gp120 with nanomolar affinity (Table 2).

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TABLE 2. The relative binding of 2G12 to BSA-(Man4)14, Man4, Man, and gp120

Taken together, the 2G12 binding data suggest that some approximation of the high-mannose clusters on the glycan shield has been achieved on BSA-(Man₄)₁₄ but that the specific epitope on gp120 has not been faithfully mimicked. It is conceivable that such mimicry is not an absolute requisite for a carbohydrate immunogen to elicit an effective gp120 cross-reactive Ab response as any Ab capable of recognizing an epitope on the glycan shield should in theory neutralize the virus via steric interference. The discrepancy in affinity for 2G12 between gp120 and BSA-(Man₄)₁₄ is likely due to differences in the conserved spacing of the glycans on gp120 constituting the 2G12 epitope compared to the more variable spacing achieved on the heterogeneous mix of BSA-(Man₄)₁₄ species (further discussion below), the latter of which may better accommodate bivalent binding of conventional Y-shaped, non-domain-exchanged Abs. Thus, the possibility that BSA-(Man₄)₁₄ may elicit anti-mannose Abs that, while somewhat different from 2G12 in specificity, are potentially cross-reactive with gp120 warranted further investigation.

Immunization with BSA-(Man₄)₁₄ elicits serum antibodies against Man₄, but these antibodies do not recognize gp120. To evaluate the immunogenicity of the BSA-(Man₄)₁₄ construct, four rabbits were each immunized with 200 µg of purified glycoconjugate or the native carrier protein (BSA). Booster injections were performed at monthly intervals, and bleeds were taken 7 to 10 days later. A total of three and four booster injections were administered to control and experimental animals, respectively. All animals remained healthy for the duration of the study, and no adverse reactions were observed that may have been indicative of autoimmune reactions to Man₄. Serum binding titers were determined against BSA-(Man₄)₁₄ and gp120_JR-CSF adsorbed directly onto ELISA wells (Fig. 2A). Binding to the irrelevant antigen Ova was also assayed as an indicator of nonspecific serum Ab binding. All assays were performed in the presence of 1% (wt/vol) BSA to absorb anti-BSA Abs. Serum from all four BSA-(Man₄)_14--immunized animals exhibited Ab titers against the Man₄ glycoconjugate. Conversely, serum from the BSA control rabbits showed little cross-reactivity with the glycoconjugate, which suggests that the BSA included in the assay buffer decreased the detection of Abs directed to the protein carrier. Thus, the reactivity observed against BSA-(Man₄)₁₄ in the experimental group may be attributed to Abs binding Man₄. Nevertheless, no serum reactivity was observed against monomeric gp120 (Fig. 2A). Protein A purification of BSA-(Man₄)₁₄ immune serum was also done to improve detection of low concentrations of (and/or low affinity) IgG and to eliminate any nonspecific inhibition by serum components; however, the results were the same (data not shown). To negate the possibility that the lack of serum reactivity may be due to defective antigen, the gp120 used was checked for binding against a number of gp120-specific monoclonal Abs including 2G12 (data not shown).

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FIG. 2. Binding of serum IgG from BSA-(Man4)14--immunized (top panel) and control (BSA)-immunized animals (bottom panel) to gp120 and Man4 glycoconjugates. (A) Binding of serially diluted sera from BSA-(Man4)14- and BSA-immunized rabbits to immobilized gp120, BSA-(Man4)14, and Ova in the presence of 1% BSA as measured by ELISA. (B) Comparison of BSA-(Man4)14 serum and BSA serum IgG binding to Man4 presented on BSA [BSA-(Man4)14] and Ova [Ova-(Man4)10]. Open symbols denote preimmune serum samples, and filled symbols denote serum samples taken after the last booster injection [bleeds 4 and 5 for BSA and BSA-(Man4)14, respectively]. OD, optical density.

To verify specific reactivity against Man₄, serum binding to an alternate Man₄ glycoconjugate, Ova-(Man₄)₁₀, was determined (Fig. 2B). The binding curves for BSA-(Man₄)₁₄ immune serum against Ova-(Man₄)₁₀ were comparable to those observed against the BSA glycoconjugate. The slight differences may be attributed to the lower copy number of Man₄ molecules and the spatial arrangement of these molecules displayed on Ova. BSA serum displayed low levels of nonspecific binding to Ova-(Man₄)₁₀, similar to that observed for BSA-(Man₄)₁₄. These observations suggest that the majority of the observed reactivity against BSA-(Man₄)₁₄ is directed against the Man₄ moieties and that the reactivity is not overtly specific to their arrangement on BSA.

Anti-mannose Abs in BSA-(Man₄)₁₄ serum recognize Man₄ but bind with less affinity to this structure in a branched context. The antiglycan specificities contained in the immune serum were further probed by in vitro competition assays with D-mannose and synthetic glycans (Man₃, Man₄, and Man₇). Man₃ is similar to Man₄ but lacks the terminal D1 12-linked mannose residue, while Man₇ is a branched bisected glycan, in which one of the two arms corresponds to the Man₄ structure. Serum (diluted 1:400) binding to BSA-(Man₄)₁₄ coated onto ELISA wells was measured in the presence of 1% BSA and 200 mM D-mannose or a 2 mM concentration of the synthetic oligomannosides. The calculated percent inhibition of serum binding attributed to each glycan, the structures of which are depicted using symbol nomenclature, is shown in Fig. 3. Note that inhibition by D-mannose must be interpreted with caution as nonspecific solvent effects may become significant at the very high concentrations of sugar required to observe a reduction in binding. Nonetheless, we observed that D-mannose did not substantially inhibit serum binding to BSA-(Man₄)₁₄, even at high molar concentrations (up to 200 mM, which is near its IC₅₀ value for 2G12). Sera from two animals were modestly inhibited by D-mannose, while no inhibition was observed for the other two sera. Man₃ inhibited binding to BSA-(Man₄)₁₄ more potently than D-mannose in three of the four sera tested, and greater than 50% inhibition was observed for two samples. Man₄ was the strongest inhibitor of all four sera, by a modest margin, reducing serum binding approximately 60 to 70%. Somewhat strikingly, Man₇ only modestly inhibited serum binding even though it contained the Man₄ structure. Taken together, these results suggest that Man₄, in the context of the neoglycoconjugate BSA-(Man₄)₁₄, elicits Abs that specifically recognize elements comprising the Man₄ structure; however, they are less able to recognize the same structure in the context of branched glycans.

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FIG. 3. Comparison of the abilities of different mannose ligands to compete with BSA-(Man4)14 immune serum IgG for binding to BSA-(Man4)14. The percent inhibition of serum IgG binding, as measured by ELISA, to BSA-(Man4)14 for D-mannose, Man3, Man4, and Man7. Serum from the fifth bleed was assayed at a 1:400 dilution. x axis, serum sample (rabbit identifier); y axis, percent inhibition of glycoconjugate binding; z axis, soluble mannoside competitor at 2 mM (or 200 mM for D-mannose, indicated by the asterisk). Symbolic representations of the ligands are included for reference.

BSA-(Man₄)₁₄ serum Abs recognize synthetic oligomannoside derivatives containing the D1 arm Man₄ structure (or fragments thereof) but not the corresponding natural, high-mannose glycans on a printed covalent glycan array. To gain further insight into the inability of the elicited anti-mannose Abs to bind gp120, a wider panel of related oligomannosides was probed on a printed glycan array. Serum was assayed at a 1:200 dilution to facilitate detection of lower-affinity interactions while minimizing nonspecific background binding. Binding of serum IgG (prebleed and final bleed) from control rabbits and BSA-(Man₄)₁₄ rabbits to structures related to Man₄ printed at 100 µM (saturating conditions) is shown in Fig. 4A. The glycans have been reordered based on size, and synthetic oligomannoside fragments are grouped together, followed by the natural, high-mannose structures isolated from RNase B. Symbolic representations of the oligosaccharide structures are depicted in Fig. 4B.

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FIG. 4. Immunogenicity of Man4 as presented on BSA-(Man4)14 in rabbits: serum IgG recognition of -D-mannose and various synthetic and naturally derived oligomannosides present on the printed glycan array, version 3.0. (A) Binding of prebleed and final bleed sera, diluted 1:200, to oligomannose glycans as measured by fluorescence (y axis). Individual glycans assayed are referenced by the glycan identification numbers on the printed array (x axis). Immune serum data are plotted directly above each glycan identification number, while preimmune serum data for the corresponding sugar are plotted above the preceding asterisk. Each symbol corresponds to a single rabbit in either the BSA group or the BSA-(Man4)14 group. For the BSA group, the symbols are as follows: green square, rabbit 18671; red triangle, 18672; open circle, 18673; and blue diamond, 18674. For the BSA-(Man4)14 group, the symbols are as follows: green square, rabbit 18667; red triangle, 18668; open circle, 18669; and blue diamond, 18670. (B) Symbolic representations of the oligosaccharide structures corresponding to the glycan identification numbers in panel A shown in the context of the larger Man9GlcNAc2 parent structure. Note that glycan 9 is not depicted as it represents the monosaccharide -D-mannose.

The anti-mannoside serum reactivity profiles indicate that BSA-(Man₄)₁₄ elicits IgG that recognizes synthetic fragments of high-mannose oligosaccharides terminating in Man12Man (glycans 191, 189, 190, 196, 313, and 314) and/or containing Man13Man motifs (glycans 191, 195, 189, 190, 196, 195, 313, and 314). Consistent with the inhibition ELISA data (Fig. 3), some impediments to binding these motifs in the context of branching were evident. For example, BSA-(Man₄)₁₄ serum generally exhibited high binding for the Man13Man motif in the biantennary glycan 195 but negligible binding to the same motif in the triantennary glycan 312. Likewise, there is a more subtle preference for binding Man₈ (glycan 313) over Man₉ (glycan 314). These tendencies are most prominent in serum 18669, which also exhibited markedly weaker binding to glycan 195 than the other BSA-(Man₄)₁₄ immune sera, suggesting poor affinity for Man13Man. Interestingly, in contrast to the distinct recognition of synthetic high-mannose fragments described above, BSA-(Man₄)₁₄ immune serum only weakly bound naturally derived oligomannose glycans (glycans 50, 198, 197, 192, 193, and 194) on the array, including Man₈GlcNAc₂ and Man₉GlcNAc₂ (glycans 193 and 194, respectively).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Usually considered a major obstacle in the development of an effective HIV-1 vaccine, the gp120 glycan shield has recently garnered attention as a potential vaccine target (59, 68). Despite the generally poor immunogenicity of heterogeneous, self glycans as discussed above, several aspects of the HIV-1 glycan shield justify exploration of its viability as a vaccine target. First, the glycan shield is completely surface exposed and readily accessible to the immune system, unlike other potential vaccine targets that are less accessible (the CD4 binding site and the membrane proximal external region) or only transiently exposed (coreceptor binding site) (17, 35, 36, 50, 55, 58, 76-78, 80). Second, the epitope for one of the few potent broadly NAbs, 2G12, is composed of a cluster of oligomannose glycans that map to this region of gp120, a clear indication that the immunological silence of the shield can be broken (8, 34, 57, 60, 65). The neutralization breadth of 2G12 indicates another attractive feature of the glycan shield, which is the presence of highly conserved oligomannose glycans that cluster together (16, 57, 60, 81). Such clustering likely forms the basis for the recognition of a glycan cluster as nonself by the 2G12 progenitor B cell(s). Indeed, no human protein is known to be as densely glycosylated as gp120. It is conceivable that other nonself epitopes may exist on this conserved region that may be exploited by a carbohydrate vaccine.

One strategy for targeting the glycan shield is to utilize 2G12 as a template for constructing an approximation of the oligomannose region recognized by the Ab that can enhance the features considered nonself. We have shown that a synthetic Man₄, corresponding to the D1 arm of Man₉GlcNAc₂, binds in the primary Ab-combining site of 2G12 and exhibits affinity comparable to the parent structure, indicating its potential as a building block for the development of a carbohydrate vaccine candidate for HIV-1 (15, 37, 77). Moreover, glycan array profiling of normal human serum indicates that the Man₄ fragment is antigenic, unlike Man₉GlcNAc₂ (5). We hypothesized that multivalent presentation of this synthetic oligomannoside on a carrier protein, via conjugation to free Lys residues, may create an approximation of the 2G12 epitope and be capable of eliciting class-switched Abs against the Man₄ determinant that may then bind gp120. A critical consideration in this strategy is the choice of carrier. BSA was chosen for an initial study as it is a commonly used scaffold for multivalent presentation of ligands for immunogenicity studies, contains numerous chemically accessible Lys residues, and has a relatively well-defined, stable three-dimensional structure (29). Although the three-dimensional structure of BSA has not been determined, chemical cross-linking of Lys residues on BSA combined with mass spectrometric analysis indicates that the spacing between eight pairs of surface-exposed Lys residues is within 24 Å, similar to the spacing estimated between the glycosylation sites on gp120 involved in 2G12 binding (16, 31).

Indeed, multivalent presentation of Man₄ on the BSA scaffold successfully increased the affinity of 2G12 for the oligosaccharide approximately 1,000-fold from mM to µM, indicating that glycan clusters may be created on the surface of a carrier protein by virtue of the number and arrangement of chemically addressable free Lys residues on the scaffold. Similar affinity boosts have been achieved utilizing different strategies for a multivalent display of oligomannose, including the synthesis of high-mannose glycopeptide fragments of gp120 and synthetic high-mannose clusters conjugated to various carrier proteins or peptides (24, 28, 33, 39, 48, 67, 69). Each strategy offers a different set of advantages in the ability to address the key issues of antigenicity versus immunogenicity. While the glycopeptide fragments may facilitate the more precise representation of the 2G12 epitope, these constructs may suffer from the same poor immunogenicity of the glycan shield. The synthesis of clusters on chemical scaffolds affords considerably more control in the spacing and presentation of sugars enabling optimization of homogenous clusters; however, the chemical scaffolds and linkers involved can themselves be immunogenic, easily distracting the immune response from the glycans of interest (48). In contrast, we would contend that precise reproduction of the specific 2G12 epitope is not required, but, rather, emulation of the dense arrangement of oligomannose on the glycan shield is the ultimate goal, where 2G12 binding is used as an estimate of this achievement. Thus, our strategy exploits the close arrangement of Lys residues on a chosen protein carrier to minimize the use of synthetic scaffold and linker elements that may divert the immune response at the expense of sacrificing some control over the final arrangement of glycans.

Although significant enhancement in binding was achieved, a large discrepancy remains between the affinity of 2G12 for gp120 and its affinity for BSA-(Man₄)₁₄. Two potential explanations may be proposed. The first is that 14 copies of Man₄, displayed on BSA, fail to provide maximum avidity for 2G12 interaction. However, Table 1 indicates that increasing the copy number of Man₄ molecules displayed on BSA beyond a certain point leads to no further increase in affinity for 2G12. The second explanation, more consistent with the above observation, is that the average spacing of glycans achieved on the BSA scaffold may not adequately represent the equivalent arrangement on gp120 for 2G12 binding. Whereas in gp120 the spacing among the two to four sugars comprising the 2G12 epitope is conserved, the location of the 14 Man₄ molecules on BSA is heterogeneous, yielding a number of potential arrangements. The affinity, therefore, of 2G12 for BSA-(Man₄)₁₄ is likely an average for the heterogeneous mixture of Man₄ arrangements found on BSA. It appears then that the affinity of BSA glycoconjugates is limited by the spacing of the glycans on the surface of BSA. The level of 2G12 binding suggests that optimal arrangements reminiscent of the glycan shield have not been made or that they are only made infrequently, a limitation inherent to this scaffold. Unfortunately, the lack of a three-dimensional structure of BSA in conjunction with the alkaline conditions used for conjugation, which can cause some protein unfolding, prevents us from deriving a reasonable prediction for the general topographic arrangement of glycans on BSA-(Man₄)₁₄.

While previous 2G12 carbohydrate immunogens were poorly immunogenic (48), BSA-(Man₄)₁₄ did elicit good anticarbohydrate titers. However, the elicited Abs bind more tightly to the synthetic Man₄ tetrasaccharide than branched oligomannosides that incorporate the Man₄ structure. Moreover, negligible binding was observed for natural oligomannose containing the D1 arm, which corresponds to Man₄, in the context of gp120 or on the glycan array. A potential explanation for the lack of recognition of gp120 may be the inability of the glycoconjugate to elicit V_H domain-exchanged Abs. While we cannot rule out this possibility, we contend that the key shortcomings of BSA-(Man₄)₁₄ lie elsewhere as the lack of domain-exchanged Abs does not adequately explain the inability of the elicited anti-mannose Abs to recognize natural oligomannosides on the glycan array.

The immunogenicity of BSA-(Man₄)₁₄ may perhaps be better understood by considering the differential conjugation of the synthetic oligomannose derivatives on a scaffold, be it a carrier protein or an activated matrix coating a glass surface (i.e., glycan array), compared to that of natural high-mannose structures. The latter are linked to Asn residues via two β-linked GlcNAc residues that form a relatively rigid, elongated structure that supports the oligomannose branches, which are then internally stabilized by a network of hydrogen bonds (74). Despite a high degree of internal flexibility within the oligomannose, the overall topology is conserved (74). When taken out of this context, the D1 arm, which is tethered via a flexible linker to BSA, may not be limited to the predominantly upright presentations supported in high-mannose structures. Rather, it is quite feasible that a greater degree of linker flexibility, which occurs in the absence of adjacent oligosaccharide chains, allows the D1 arm to lie more parallel to the protein surface, a situation likely encountered on BSA-(Man₄)₁₄ [and probably Ova-(Man₄)₁₀], given its heterogeneous nature. As these orientations are not usually adopted by natural high-mannose structures on self proteins, they may be immunodominant and elicit Abs specific to Man₄ presented in this way. One possible explanation for the recognition profile of the elicited Abs against the Man₄ conjugate that is consistent with this scenario is that their Ab combining sites are groove-like compared to the deep pocket in 2G12 that binds the tips of Man₄ (i.e., an end binder) in natural high-mannose N-glycans on gp120. Both D-mannose and Man₃ would be incapable of filling the entire binding surface, while branched sugars, e.g., Man₇ and Man₉, would be sterically hindered by the additional arm(s). Perhaps Abs with these antigen binding sites readily recognize synthetic high-mannose but not natural high-mannose structures because the linker-dependent presentations described above are also adopted on the printed covalent glycan array. In addition, increased positional fluctuations may disrupt the hydrogen bonding network between the antennae of branched synthetic oligomannosides (e.g., Man₈ and Man₉), also facilitating Ab binding (26).

In summary, the characterization of BSA-(Man₄)₁₄ as both an antigen for 2G12 and an immunogen for eliciting anti-mannose Abs to target the HIV-1 glycan shield has provided insights into the complex relationship between antigenicity and immunogenicity as it relates to essentially self glycans comprising this surface of gp120. We reiterate the importance of multivalency for 2G12 binding to its minimal epitope and have shown that multivalent presentation of Man₄, a synthetic derivative of high-mannose glycans representing the D1 arm, can be immunogenic and elicit mannose-specific IgG capable of recognizing Man₄ presented on more than one scaffold. However, the elicited Abs do not recognize the D1 arm within a natural oligomannoside context, which suggests potential differences in the presentation and accessibility of synthetic compared to natural oligomannosides. These results are also consistent with the low affinity of 2G12 for binding to BSA-(Man₄)₁₄ compared to gp120. We reason that these differences in presentation may stem from the flexibility conferred upon the glycans by the synthetic linker, and, while one cannot dispose of the linker, it may be possible to minimize its effects. We infer from this initial study that the presentation of branched and/or densely clustered glycans is likely required to better mimic the glycan shield recognized by 2G12 so as to elicit Abs that bind gp120. Branched structures including both the D1 and D3 arms, such as Man₈ and Man₉, may also be more advantageous as 2G12 has been shown to bind both of these motifs (15). To achieve a more controlled and denser display, we are currently exploring the use of noninfectious, virus-like particles with icosahedral symmetry and synthetic oligodendrons as scaffolds. A greater understanding of how the immune system recognizes oligomannose neoglycoconjugates compared to recognition of natural high-mannose glycoproteins like gp120 is paramount to the design of an effective carbohydrate vaccine against the HIV-1 glycan shield. The challenge likely lies not in eliciting Abs to exactly the same carbohydrate determinant (Man₄) as 2G12 but, rather, in eliciting Abs that recognize the nonreducing termini of 12-linked mannose residues within oligomannose clusters in an "end-binding" manner similar to the recognition mode of 2G12.

 
We greatly appreciate the gift of 2G12 from Gabriela Steigler and Herman Katinger (Polymun Scientific, Vienna, Austria, and University of Natural Resources and Applied Life Sciences, Vienna, Austria). We also thank the CFG, especially James Paulson, Julia Busch, Julia Hoffmann, and Ryan McBride, for enabling us to perform the glycan array analyses and for helpful discussion. We also thank James C. Paulson for helpful scientific discussion and critical reading of the manuscript.

The work was supported by the Natural Sciences and Engineering Research Council of Canada (to R.D.A.), the International AIDS Vaccine Initiative Neutralizing Antibody Consortium (to D.R.B., I.A.W., C.N.S., and C.-H.W.), and the National Institute of Health grants GM44154 (to C.H.W.), GM46192 (to I.A.W.), AI33292, and AI060425 (to D.R.B.).

 
* Corresponding author. Mailing address: The Scripps Research Institute, Department of Immunology and Microbial Science, IMM-2, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-9298. Fax: (858) 784-8360. E-mail: burton{at}scripps.edu

Published ahead of print on 23 April 2008.

Present address: Intertek Testing Services Shenzhen Ltd., 7/F Shekou Technology Main Building, Industrial 7th Road, Shekou, Nanshan District, Shenzhen, China 518067.

Present address: Research Center for Materials Science, Nagoya University, Nagoya 464-8602, Japan.

Present address: Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Blegdamsvej 3B, Copenhagen N 2200, Denmark.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie, L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou, and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med. 6:200-206.[CrossRef][Medline]
2
2. Barbas, C. F., III, T. A. Collet, W. Amberg, P. Roben, J. M. Binley, D. Hoekstra, D. Cababa, T. M. Jones, R. A. Williamson, G. R. Pilkington, et al. 1993. Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. J. Mol. Biol. 230:812-823.[CrossRef][Medline]
3
3. Beirnaert, E., S. De Zutter, W. Janssens, and G. van der Groen. 2001. Potent broad cross-neutralizing sera inhibit attachment of primary HIV-1 isolates (groups M and O) to peripheral blood mononuclear cells. Virology 281:305-314.[CrossRef][Medline]
4
4. Binley, J. M., T. Wrin, B. Korber, M. B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C. J. Petropoulos, and D. R. Burton. 2004. Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J. Virol. 78:13232-13252.[Abstract/Free Full Text]
5
5. Blixt, O., S. Head, T. Mondala, C. Scanlan, M. E. Huflejt, R. Alvarez, M. C. Bryan, F. Fazio, D. Calarese, J. Stevens, N. Razi, D. J. Stevens, J. J. Skehel, I. van Die, D. R. Burton, I. A. Wilson, R. Cummings, N. Bovin, C. H. Wong, and J. C. Paulson. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101:17033-17038.[Abstract/Free Full Text]
6
6. Bolmstedt, A., S. Sjolander, J. E. Hansen, L. Akerblom, A. Hemming, S. L. Hu, B. Morein, and S. Olofsson. 1996. Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response. J. Acquir. Immune Defic. Syndr. Hum. Retrovir. 12:213-220.[Medline]
7
7. Bryan, M. C., F. Fazio, H. K. Lee, C. Y. Huang, A. Chang, M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton, I. A. Wilson, and C. H. Wong. 2004. Covalent display of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640-8641.[CrossRef][Medline]
8
8. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retrovir. 10:359-369.[Medline]
9
9. Burton, D. R. 2002. Antibodies, viruses and vaccines. Nat. Rev. Immunol. 2:706-713.[CrossRef][Medline]
10
10. Burton, D. R. 1997. A vaccine for HIV type 1: the antibody perspective. Proc. Natl. Acad. Sci. USA 94:10018-10023.[Abstract/Free Full Text]
11
11. Burton, D. R., C. F. Barbas III, M. A. Persson, S. Koenig, R. M. Chanock, and R. A. Lerner. 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. USA 88:10134-10137.[Abstract/Free Full Text]
12
12. Burton, D. R., R. C. Desrosiers, R. W. Doms, W. C. Koff, P. D. Kwong, J. P. Moore, G. J. Nabel, J. Sodroski, I. A. Wilson, and R. T. Wyatt. 2004. HIV vaccine design and the neutralizing antibody problem. Nat. Immunol. 5:233-236.[CrossRef][Medline]
13
13. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L. Nara, et al. 1994. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266:1024-1027.[Abstract/Free Full Text]
14
14. Burton, D. R., R. L. Stanfield, and I. A. Wilson. 2005. Antibody vs. HIV in a clash of evolutionary titans. Proc. Natl. Acad. Sci. USA 102:14943-14948.[Abstract/Free Full Text]
15
15. Calarese, D. A., H. K. Lee, C. Y. Huang, M. D. Best, R. D. Astronomo, R. L. Stanfield, H. Katinger, D. R. Burton, C. H. Wong, and I. A. Wilson. 2005. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. Natl. Acad. Sci. USA 102:13372-13377.[Abstract/Free Full Text]
16
16. Calarese, D. A., C. N. Scanlan, M. B. Zwick, S. Deechongkit, Y. Mimura, R. Kunert, P. Zhu, M. R. Wormald, R. L. Stanfield, K. H. Roux, J. W. Kelly, P. M. Rudd, R. A. Dwek, H. Katinger, D. R. Burton, and I. A. Wilson. 2003. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300:2065-2071.[Abstract/Free Full Text]
17
17. Chen, B., E. M. Vogan, H. Gong, J. J. Skehel, D. C. Wiley, and S. C. Harrison. 2005. Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 433:834-841.[CrossRef][Medline]
18
18. Conley, A. J., M. K. Gorny, J. A. Kessler II, L. J. Boots, M. Ossorio-Castro, S. Koenig, D. W. Lineberger, E. A. Emini, C. Williams, and S. Zolla-Pazner. 1994. Neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-V3 monoclonal antibody, 447-52D. J. Virol. 68:6994-7000.[Abstract/Free Full Text]
19
19. Conley, A. J., J. A. Kessler II, L. J. Boots, J. S. Tung, B. A. Arnold, P. M. Keller, A. R. Shaw, and E. A. Emini. 1994. Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody. Proc. Natl. Acad. Sci. USA 91:3348-3352.[Abstract/Free Full Text]
20
20. Cutalo, J. M., L. J. Deterding, and K. B. Tomer. 2004. Characterization of glycopeptides from HIV-I(SF2) gp120 by liquid chromatography mass spectrometry. J. Am. Soc. Mass Spectrom. 15:1545-1555.[CrossRef][Medline]
21
21. Donners, H., B. Willems, E. Beirnaert, R. Colebunders, D. Davis, and G. van der Groen. 2002. Cross-neutralizing antibodies against primary isolates in African women infected with HIV-1. AIDS 16:501-503.[CrossRef][Medline]
22
22. Douek, D. C., P. D. Kwong, and G. J. Nabel. 2006. The rational design of an AIDS vaccine. Cell 124:677-681.[CrossRef][Medline]
23
23. D'Souza, M. P., D. Livnat, J. A. Bradac, S. H. Bridges, et al. 1997. Evaluation of monoclonal antibodies to human immunodeficiency virus type 1 primary isolates by neutralization assays: performance criteria for selecting candidate antibodies for clinical trials. J. Infect. Dis. 175:1056-1062.[Medline]
24
24. Dudkin, V. Y., M. Orlova, X. Geng, M. Mandal, W. C. Olson, and S. J. Danishefsky. 2004. Toward fully synthetic carbohydrate-based HIV antigen design: on the critical role of bivalency. J. Am. Chem. Soc. 126:9560-9562.[CrossRef][Medline]
25
25. Ferrantelli, F., R. A. Rasmussen, K. A. Buckley, P. L. Li, T. Wang, D. C. Montefiori, H. Katinger, G. Stiegler, D. C. Anderson, H. M. McClure, and R. M. Ruprecht. 2004. Complete protection of neonatal rhesus macaques against oral exposure to pathogenic simian-human immunodeficiency virus by human anti-HIV monoclonal antibodies. J. Infect. Dis. 189:2167-2173.[CrossRef][Medline]
26
26. Fromme, R., Z. Katiliene, B. Giomarelli, F. Bogani, J. Mc Mahon, T. Mori, P. Fromme, and G. Ghirlanda. 2007. A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity. Biochemistry 46:9199-9207.[CrossRef][Medline]
27
27. Gauduin, M. C., P. W. Parren, R. Weir, C. F. Barbas, D. R. Burton, and R. A. Koup. 1997. Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nat. Med. 3:1389-1393.[CrossRef][Medline]
28
28. Geng, X., V. Y. Dudkin, M. Mandal, and S. J. Danishefsky. 2004. In pursuit of carbohydrate-based HIV vaccines, part 2: the total synthesis of high-mannose-type gp120 fragments—evaluation of strategies directed to maximal convergence. Angew. Chem. Int. Ed. Engl. 43:2562-2565.[CrossRef]
29
29. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
30
30. Hofmann-Lehmann, R., J. Vlasak, R. A. Rasmussen, B. A. Smith, T. W. Baba, V. Liska, F. Ferrantelli, D. C. Montefiori, H. M. McClure, D. C. Anderson, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, H. Katinger, G. Stiegler, L. A. Cavacini, M. R. Posner, T. C. Chou, J. Andersen, and R. M. Ruprecht. 2001. Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge. J. Virol. 75:7470-7480.[Abstract/Free Full Text]
31
31. Huang, B. X., H. Y. Kim, and C. Dass. 2004. Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry. J. Am. Soc. Mass Spectrom. 15:1237-1247.[CrossRef][Medline]
32
32. Johnston, M. I., and A. S. Fauci. 2007. An HIV vaccine—evolving concepts. N. Engl. J. Med. 356:2073-2081.[Free Full Text]
33
33. Krauss, I. J., J. G. Joyce, A. C. Finnefrock, H. C. Song, V. Y. Dudkin, X. Geng, J. D. Warren, M. Chastain, J. W. Shiver, and S. J. Danishefsky. 2007. Fully synthetic carbohydrate HIV antigens designed on the logic of the 2G12 antibody. J. Am. Chem. Soc. 129:11042-11044.[CrossRef][Medline]
34
34. Kunert, R., F. Ruker, and H. Katinger. 1998. Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS Res. Hum. Retrovir. 14:1115-1128.[Medline]
35
35. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648-659.[CrossRef][Medline]
36
36. Kwong, P. D., R. Wyatt, Q. J. Sattentau, J. Sodroski, and W. A. Hendrickson. 2000. Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. J. Virol. 74:1961-1972.[Abstract/Free Full Text]
37
37. Lee, H. K., C. N. Scanlan, C. Y. Huang, A. Y. Chang, D. A. Calarese, R. A. Dwek, P. M. Rudd, D. R. Burton, I. A. Wilson, and C. H. Wong. 2004. Reactivity-based one-pot synthesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody. Angew. Chem. Int. Ed. Engl. 43:1000-1003.[CrossRef]
38
38. Letvin, N. L. 2006. Progress and obstacles in the development of an AIDS vaccine. Nat. Rev. Immunol. 6:930-939.[CrossRef][Medline]
39
39. Li, H., and L. X. Wang. 2004. Design and synthesis of a template-assembled oligomannose cluster as an epitope mimic for human HIV-neutralizing antibody 2G12. Org. Biomol. Chem. 2:483-488.[CrossRef][Medline]
40
40. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X. Wu, G. M. Shaw, M. Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med. 13:1032-1034.[CrossRef][Medline]
41
41. Mandal, M., V. Y. Dudkin, X. Geng, and S. J. Danishefsky. 2004. In pursuit of carbohydrate-based HIV vaccines, part 1: the total synthesis of hybrid-type gp120 fragments. Angew. Chem. Int. Ed. Engl. 43:2557-2561.[CrossRef]
42
42. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb, H. Katinger, and D. L. Birx. 1999. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73:4009-4018.[Abstract/Free Full Text]
43
43. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter, C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6:207-210.[CrossRef][Medline]
44
44. McCaffrey, R. A., C. Saunders, M. Hensel, and L. Stamatatos. 2004. N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies. J. Virol. 78:3279-3295.[Abstract/Free Full Text]
45
45. McMichael, A. J. 2006. HIV vaccines. Annu. Rev. Immunol. 24:227-255.[CrossRef][Medline]
46
46. Mehandru, S., B. Vcelar, T. Wrin, G. Stiegler, B. Joos, H. Mohri, D. Boden, J. Galovich, K. Tenner-Racz, P. Racz, M. Carrington, C. Petropoulos, H. Katinger, and M. Markowitz. 2007. Adjunctive passive immunotherapy in human immunodeficiency virus type 1-infected individuals treated with antiviral therapy during acute and early infection. J. Virol. 81:11016-11031.[Abstract/Free Full Text]
47
47. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647.[Abstract/Free Full Text]
48
48. Ni, J., H. Song, Y. Wang, N. M. Stamatos, and L. X. Wang. 2006. Toward a carbohydrate-based HIV-1 vaccine: synthesis and immunological studies of oligomannose-containing glycoconjugates. Bioconjug. Chem. 17:493-500.[CrossRef][Medline]
49
49. Nishimura, Y., T. Igarashi, N. Haigwood, R. Sadjadpour, R. J. Plishka, A. Buckler-White, R. Shibata, and M. A. Martin. 2002. Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies. J. Virol. 76:2123-2130.[Abstract/Free Full Text]
50
50. Pantophlet, R., and D. R. Burton. 2006. gp120: target for neutralizing HIV-1 antibodies. Annu. Rev. Immunol. 24:739-769.[CrossRef][Medline]
51
51. Parren, P. W., H. J. Ditzel, R. J. Gulizia, J. M. Binley, C. F. Barbas III, D. R. Burton, and D. E. Mosier. 1995. Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site. AIDS 9:F1-F6.[Medline]
52
52. Parren, P. W., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng-Mayer, J. P. Moore, and D. R. Burton. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J. Virol. 75:8340-8347.[Abstract/Free Full Text]
53
53. Ratner, D. M., and P. H. Seeberger. 2007. Carbohydrate microarrays as tools in HIV glycobiology. Curr. Pharm. Des. 13:173-183.[CrossRef][Medline]
54
54. Richman, D. D., T. Wrin, S. J. Little, and C. J. Petropoulos. 2003. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc. Natl. Acad. Sci. USA 100:4144-4149.[Abstract/Free Full Text]
55
55. Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R. Burton. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 68:4821-4828.[Abstract/Free Full Text]
56
56. Rudd, P. M., and R. A. Dwek. 1997. Glycosylation: heterogeneity and the 3D structure of proteins. Crit. Rev. Biochem. Mol. Biol. 32:1-100.[Medline]
57
57. Sanders, R. W., M. Venturi, L. Schiffner, R. Kalyanaraman, H. Katinger, K. O. Lloyd, P. D. Kwong, and J. P. Moore. 2002. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 76:7293-7305.[Abstract/Free Full Text]
58
58. Sattentau, Q. J., and J. P. Moore. 1995. Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer. J. Exp. Med. 182:185-196.[Abstract/Free Full Text]
59
59. Scanlan, C. N., J. Offer, N. Zitzmann, and R. A. Dwek. 2007. Exploiting the defensive sugars of HIV-1 for drug and vaccine design. Nature 446:1038-1045.[CrossRef][Medline]
60
60. Scanlan, C. N., R. Pantophlet, M. R. Wormald, E. Ollmann Saphire, R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R. Burton. 2002. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of 12 mannose residues on the outer face of gp120. J. Virol. 76:7306-7321.[Abstract/Free Full Text]
61
61. Shibata, R., T. Igarashi, N. Haigwood, A. Buckler-White, R. Ogert, W. Ross, R. Willey, M. W. Cho, and M. A. Martin. 1999. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat. Med. 5:204-210.[CrossRef][Medline]
62
62. Thomas, R., S. I. Patenaude, C. R. MacKenzie, R. To, T. Hirama, N. M. Young, and S. V. Evans. 2002. Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity. J. Biol. Chem. 277:2059-2064.[Abstract/Free Full Text]
63
63. Trkola, A., H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard. 2005. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat. Med. 11:615-622.[CrossRef][Medline]
64
64. Trkola, A., A. B. Pomales, H. Yuan, B. Korber, P. J. Maddon, G. P. Allaway, H. Katinger, C. F. Barbas III, D. R. Burton, and D. D. Ho. 1995. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J. Virol. 69:6609-6617.[Abstract]
65
65. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70:1100-1108.[Abstract]
66
66. Vliegenthart, J. F. 2006. Carbohydrate based vaccines. FEBS Lett. 580:2945-2950.[CrossRef][Medline]
67
67. Wang, J., H. Li, G. Zou, and L. X. Wang. 2007. Novel template-assembled oligosaccharide clusters as epitope mimics for HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study. Org. Biomol. Chem. 5:1529-1540.[CrossRef][Medline]
68
68. Wang, L. X. 2006. Toward oligosaccharide- and glycopeptide-based HIV vaccines. Curr. Opin. Drug Discov. Devel. 9:194-206.[Medline]
69
69. Wang, L. X., J. Ni, S. Singh, and H. Li. 2004. Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem. Biol. 11:127-134.[CrossRef][Medline]
70
70. Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307-312.[CrossRef][Medline]
71
71. Weis, W. I., and K. Drickamer. 1996. Structural basis of lectin-carbohydrate recognition. Annu. Rev. Biochem. 65:441-473.[CrossRef][Medline]
72
72. Wilson, I. A., and R. L. Stanfield. 1995. A Trojan horse with a sweet tooth. Nat. Struct. Biol. 2:433-436.[CrossRef][Medline]
73
73. Woods, R. J., C. J. Edge, and R. A. Dwek. 1994. Protein surface oligosaccharides and protein function. Nat. Struct. Biol. 1:499-501.[CrossRef][Medline]
74
74. Woods, R. J., A. Pathiaseril, M. R. Wormald, C. J. Edge, and R. A. Dwek. 1998. The high degree of internal flexibility observed for an oligomannose oligosaccharide does not alter the overall topology of the molecule. Eur. J. Biochem. 258:372-386.[Medline]
75
75. World Health Organization. 2007. AIDS epidemic update: December 2007. Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization, Geneva, Switzerland.
76
76. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705-711.[CrossRef][Medline]
77
77. Wyatt, R., and J. Sodroski. 1998. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280:1884-1888.[Abstract/Free Full Text]
78
78. Zanetti, G., J. A. Briggs, K. Grunewald, Q. J. Sattentau, and S. D. Fuller. 2006. Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog. 2:e83.[CrossRef][Medline]
79
79. Zhang, P. F., F. Cham, M. Dong, A. Choudhary, P. Bouma, Z. Zhang, Y. Shao, Y. R. Feng, L. Wang, N. Mathy, G. Voss, C. C. Broder, and G. V. Quinnan, Jr. 2007. Extensively cross-reactive anti-HIV-1 neutralizing antibodies induced by gp140 immunization. Proc. Natl. Acad. Sci. USA 104:10193-10198.[Abstract/Free Full Text]
80
80. Zhu, P., J. Liu, J. Bess, Jr., E. Chertova, J. D. Lifson, H. Grise, G. A. Ofek, K. A. Taylor, and K. H. Roux. 2006. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441:847-852.[CrossRef][Medline]
81
81. Zhu, X., C. Borchers, R. J. Bienstock, and K. B. Tomer. 2000. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells. Biochemistry 39:11194-11204.[CrossRef][Medline]
82
82. Zwick, M. B., A. F. Labrijn, M. Wang, C. Spenlehauer, E. O. Saphire, J. M. Binley, J. P. Moore, G. Stiegler, H. Katinger, D. R. Burton, and P. W. Parren. 2001. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J. Virol. 75:10892-10905.[Abstract/Free Full Text]

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Journal of Virology, July 2008, p. 6359-6368, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00293-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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