Variable Loop Glycan Dependency of the Broad ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 2010, p. 10510–10521 0022-538X/10/$12.00 doi:10.1128/JVI.00552-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 84, No. 20

Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16䌤 Katie J. Doores1,2 and Dennis R. Burton1,2* Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,1 and Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Boston, Massachusetts 021142 Received 12 March 2010/Accepted 26 July 2010

The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1ⴚ/ⴚ) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies. It is argued that an effective HIV vaccine should include a component that induces a broadly neutralizing antibody response (2, 3, 21, 25, 32, 37, 39, 54). The key target for broadly neutralizing HIV antibodies is the envelope spike, which consists of a compact, metastable heterodimeric trimer of the glycoproteins gp120 and gp41 (43, 62). gp120 is one of the most heavily glycosylated proteins known, with up to 50% of its mass arising from carbohydrates attached to roughly 25 N-linked glycosylation sites (31) determined by the NXT/S consensus sequence (where X can be any amino acid except Pro) (1). Glycosylation significantly impacts the folding and conformation of envelope spikes, thus affecting antigenicity and immunogenicity (30, 35). Carbohydrates are generally poorly immunogenic, and the dense covering of glycans is often referred to as the “silent face” or “glycan shield” (58). The glycans have also been suggested to have an important role in viral transmission through interaction with lectins, in particular the C-type lectin DC-SIGN, which is found on the surfaces of dendritic cells and is thought to aid the transport of virus to anatomical sites rich in CD4⫹ T cells, such as lymph nodes (8, 16). Although the positioning of N-linked protein glycosylation is encoded by the protein sequence (1), the type of glycan displayed (high mannose, hybrid, or complex) is not under direct genetic control but is determined by the three-dimensional structure of a protein and its interaction with the biosynthetic cellular environment, including accessibility to glycan-processing enzymes (50). For example, highly clustered glycans prevent access of the processing enzymes, leading to high-man* Corresponding author. Mailing address: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037. Phone: (858) 784-9298. Fax: (858) 784-8360. E-mail: burton @scripps.edu. 䌤 Published ahead of print on 4 August 2010.

nose-type glycans being displayed (6, 23). Therefore, the glycosylation of recombinant HIV envelope proteins can vary significantly depending on the protein sequence, structure, and the cell in which they are expressed (50). Although the positions of many glycans are relatively conserved between isolates and clades (60), there can be variation in the occupancy and precise nature of the glycans displayed at these positions on recombinant envelope (7, 17–19, 61). However, we have recently observed major differences between the glycosylation of recombinant envelope proteins and envelope expressed on the virion surface, with the latter being dominated by Man5-9GlcNAc2 oligomannose glycans (9). Nevertheless, significant glycan heterogeneity remains on the virion surface. Recently, two new neutralizing antibodies, PG9 and PG16, were isolated from an African clade A-infected donor and shown to be both broad and potent (56). From a panel of 162 viruses, PG9 neutralized 127 and PG16 neutralized 119 viruses at a median potency that exceeded that of the broadly neutralizing antibodies—2G12, b12, 2F5, and 4E10—by about an order of magnitude. In a TZM-bl neutralization assay, PG9 has been shown to neutralize 87% of a panel of 82 viruses (M. Seaman, unpublished data). Both PG9 and PG16 show preferential trimer binding and interact with an epitope formed from conserved regions of the V1/V2 and V3 variable loops. Mutation of N160, an N-linked glycosylation site in the V2 loop, completely abolishes PG9 and PG16 neutralization, suggesting the N160 glycan is important in forming the PG9 and PG16 epitope. Further, PG9 shows significant binding to monomeric gp120 DU422 and treatment of the glycoprotein with Endo H (removing high-mannose glycans) results in significant reduction in antibody binding. Occasionally, neutralization of some pseudoviruses by PG16 in particular has revealed an unusual neutralization profile with a shallow slope and plateaus at ⬍100%. We hypothesized that this unusual neutral-

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ization profile may be related to antibody sensitivity to glycosylation and, more specifically, could be due to glycan profile or partial glycosylation at critical sites. We show here that loss of any one of several glycosylation sites in the V1, V2, and V3 loops has significant effects on the sensitivity of pseudovirus to neutralization by PG9 and PG16. Generating pseudovirus in the presence of various glycosidase inhibitors also has notable effects on antibody neutralization. We show that some of these effects are isolate dependent and others are universal and can be used to help identify the presence of PG9 and PG16-like antibodies in the serum of HIV1-infected patients (57). For some isolates displaying aberrant neutralization profiles as described above, we found that changing the glycan profile on the HIV-1 trimer using glycosidase inhibitors or a mutant cell line resulted in higher neutralization plateaus and neutralization profiles with the more usual sigmoidal shape. Changes in sensitivity to neutralization were also observed for some but not all isolates. The antibodygp120 interaction was not inhibited by sugar monosaccharides found in glycans on the HIV envelope. The results suggest PG9 and PG16 recognize a conformational epitope that is dependent on the glycosylation at specific variable loop N-linked glycosylation sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.

MATERIALS AND METHODS Competition enzyme-linked immunosorbent assay (ELISA) with gp120. Determination of monoclonal antibody (MAb) binding inhibition by monosaccharides was performed as previously described (51). Briefly, 250 ng of gp120DU422 was coated onto flat-bottom microtiter plates (Costar type 3690; Corning, Inc.) at 4°C overnight. All subsequent steps were performed at room temperature. The plates were washed five times with phosphate-buffered saline–0.05% Tween 20 (PBS-T) and then blocked with 3% bovine serum albumin (BSA; 100 ␮l/well) for 1 h. The wells were emptied, and 2G12 (0.2 ␮g/ml) and PG9 (2 ␮g/ml) diluted with 1% BSA–0.02% Tween 20 (PBS-BT) were added in the presence of serial dilutions of monosaccharides, followed by incubation for 2 h. After washing, antibody binding was probed with alkaline phosphatase-conjugated goat antihuman IgG Fc antibody (Jackson) diluted to 1:1,000 in PBS-BT for 1 h. The wells were washed, and the bound secondary antibody was visualized with p-nitrophenol phosphate substrate (Sigma). Mannose competition with HIV Env-transfected cells. HIV env transfected 293T cells were fixed with 1% formaldehyde solution. Antibody was added in the presence of serial dilutions of mannose and incubated for 1 h at room temperature. Cells were washed and blocked with fetal bovine serum (30 min, 4°C). Cells were washed, and bound antibody was probed with goat anti-human-Fc␥phycoerythrin (2.5 ␮g/ml, 30 min, 4°C). Binding was analyzed by flow cytometry, and binding curves were generated by plotting the mean fluorescence intensity of the antigen as a function of antibody concentration. A FACSarray plate reader (BD Biosciences) was used for flow cytometry, and FlowJo for Mac software (v8) was used for data analysis. Generation of pseudovirus. Pseudovirus was generated in HEK 293T cells as described previously (33, 40). Briefly, 293T cells were transfected with plasmids carrying the reporter gene expressing the virus backbone PSG-3⌬env and the functional envelope clone at a ratio of 2:1 using Fugene (Roche) according to the manufacturer’s instructions. Virus supernatants were harvested after 3 days. Glycosidase inhibitors were added at the time of transfection and were used at the following concentrations: 25 ␮M kifunensine, 20 ␮M swainsonine, and 2 mM N-butyldeoxynojirimycin (NB-DNJ). Neutralization assay. Neutralization activity of antibodies against pseudovirus in TZM-bl cells was determined as described previously (33, 40). Briefly, TZM-bl cells were seeded in a 96-well flat-bottom plate and infected with pseudovirus in the presence of inhibitors (200 ␮l, total volume). Viruses were preincubated with the antibody for 1 h at 37°C. Luciferase reporter gene expression was quantified 72 h after infection upon lysis and the addition of luciferase substrate (Promega).

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Plasmid constructs and mutagenesis. All envelope sequences were used in the pSVIII plasmid (22). Alanine point mutations were made by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) in the pSVIII plasmid (22, 44, 63). All mutations were verified by DNA sequence analysis (Eton Bioscience, San Diego, CA). Full envelope sequencing was carried out for all clones. Cell surface binding. Serial dilutions of antibodies were added to HIV-1 Env-transfected 293T cells, followed by incubation for 1 h at room temperature. The procedure described above was then followed (56). Mixed trimer experiment. 293T cells were cotransfected with PSG-3⌬env and wild-type or mutant envelope plasmids at various ratios. Virus was harvested after 72 h and used in neutralization assays as described above. Measurement of infectivity of virus mutants. Virus stocks were titrated on TZM-bl cells according to the method of Li et al. (33), and the TCID50 was calculated as described previously (24, 46). The p24 of the virus stocks was determined by using the Aalto p24 ELISA protocol (protocol 2), and the infectivity is reported as 50% tissue culture infective dose/ng of p24.

RESULTS Competition with monosaccharides. As described previously (56), treatment of gp120DU422, one of the few monomeric gp120 preparations to show measurable affinity for PG9, with Endo H to remove high-mannose glycans significantly decreased antibody binding (56). This suggested the possibility that mannose residues might form part of the PG9 epitope by contacting the binding site directly. To investigate this further, a competition ELISA was performed between gp120DU422 and PG9 in the presence of various concentrations of the monosaccharides D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine, and N-(2-acetyl)-neuraminic acid (sialic acid). None showed inhibition of the PG9-gp120 interaction (Fig. 1A) under conditions for which mannose has been shown to inhibit the binding of the anti-glycan antibody 2G12 to gp120 (Fig. 1B) (4). In a second monosaccharide competition format, HEK 293T cells expressing JRCSF HIV-1 envelope trimer were fixed with 1% formaldehyde solution, and the ability of mannose to inhibit PG9 or PG16 binding was assessed. No inhibition of PG9 or PG16 binding was observed under conditions (1 M mannose) for which 2G12 binding was greatly reduced (Fig. 1C). Thus, in the context of the HIV trimer, mannose is unable to compete with binding of PG9 and PG16. Alanine scanning of N-linked glycosylation sites in envelope sequences. The N-linked glycosylation site at position N160 has previously been shown to be critical for PG9 and PG16 neutralization (56). Amino acid substitution to remove this Nlinked glycosylation site renders the virus insensitive to PG9 and PG16 neutralization. To determine whether other specific N-linked glycosylation sites were critical for expression of the PG9 and PG16 epitopes, alanine mutants were generated for several isolates of HIV-1. We focused on N-linked glycosylation sites in the V1, V2, and V3 loops of the isolates JR-CSF, JR-FL (E168K) variant, 92RW020, and SF162 (K160N) variant. We generated pseudoviruses with single alanine substitutions in which potential N-linked glycosylation sites were removed by replacing Asn with Ala and then determined the neutralizing activity of PG9 and PG16 against these viruses. 92RW020 and JR-FL E168K were chosen due to the shallow neutralization profile, and SF162 K160N were chosen due to less than 100% neutralization plateaus. The JR-FL (E168K) variant and SF162 (K160N) variants were used rather than the parent JR-FL and SF162 viruses since the variants but not the parent viruses are sensitive to PG9 and PG16 neutralization (56). The infectivity of the alanine mutants was not compro-

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FIG. 1. Monosaccharide competition for MAb binding to Env. (A) Monosaccharide competition of PG9 binding to gp120DU422 in ELISA. (B) Monosaccharide competition of 2G12 binding to gp120JR-FL in ELISA. (C) Mannose competition of MAbs to fixed JR-CSF Env-expressing 293T cells by flow cytometry.

mised significantly compared to the wild-type virus (see Table S1 in the supporting information [http://www.scripps.edu/ims /burton/supplemental/Doores_JVI_2010A.pdf]). For the JR-CSF isolate, loss of the glycan at position N156 was associated with a large decrease in potency for both PG9 and PG16 (280- and 1,500-fold, respectively). However, loss of other glycosylation sites in the V1, V2, or V3 loops showed much lower, largely insignificant effects on neutralization potency (Fig. 2A and B). For the JR-FL (E168K) variant, PG9 and PG16 neutralization was found to be sensitive to the loss of several potential N-linked glycosylation sites (Fig. 2C and D). In contrast to wild-type JR-CSF, which has the typical sigmoidal shaped curve for PG9 and PG16 neutralization, the profile for wildtype JR-FL E168K displays a shallow sloping curve for PG16, and 100% neutralization is not reached by both PG9 and PG16 (compare Fig. 2A and B to Fig. 2C and D). Several substitutions in glycan consensus sequences in the JR-FL E168K backbone to generate sequences found in JR-CSF resulted in changes to the neutralization profile of PG9 and PG16 (Fig. 2C and D). In particular, an N189A substitution in the JR-FL variant that results in loss of a potential N-linked glycosylation site gave rise to a neutralization curve for both PG9 and PG16 that was sigmoidal and resembled that of JR-CSF (Fig. 2C and D). Increases in the neutralization potency of PG16 but still incomplete neutralization were seen with substitutions N295A, N332A, and N339A in JR-FL E168K (Fig. 2D). For PG9, decreases in neutralization potency and incomplete neutralization were observed with multiple glycan knockouts: N135A, N142S, N188A, N295A, N332A, and N339A (Fig. 2C). For the isolates 92RW020 and SF162 K160N, similarly com-

plex isolate and antibody-dependent effects were observed with changes in N-linked glycosylation (Fig. S1 in the supporting information [http://www.scripps.edu/ims/burton/supplemental /Doores_JVI_2010A.pdf]). PG16 was found to be more sensitive to the loss of N-linked glycans than PG9. Effect of glycosidase inhibitors on PG9 and PG16 neutralization activity. We hypothesized that the shallow sloping neutralization profile and low neutralization plateau observed with some HIV-1 isolates may be associated with glycan heterogeneity and the type of glycan present on the HIV trimer. To control the glycans displayed on the trimer in the context of pseudovirus and thereby investigate the role of glycosylation on neutralizing activity of PG9 and PG16, two approaches were adopted: the use of glycosidase inhibitors and the use of glycosidase-deficient cell lines (GnT1⫺/⫺). The glycosidase inhibitor approach is considered first. Nlinked glycosylation occurs at asparagine residues that are part of the consensus sequence NXT/S (where X can be any amino acid except proline). Initially, Glc3Man9GlcNAc2 is transferred from dolichol phosphate in the endoplasmic reticulum (ER) by oligosaccharyltransferase (Fig. 3) (29). This glycan is then trimmed by ER-␣-glucosidases I and II, which remove three terminal glucose residues, leaving Man9GlcNAc2. This glycan is then trimmed by ER-mannosidase enzymes, and the resulting Man5GlcNAc2 glycan can undergo further enzymatic remodeling in the Golgi body to give either hybrid or complex glycans. There have been several studies investigating the effect of glycosidase inhibitors on viral replication and infectivity in an attempt to generate therapeutics against HIV (20, 41). ␣-Glucosidase inhibitors (e.g., N-butyldeoxynojirimycin [NBDNJ] and castanospermine) are potent inhibitors of HIV rep-

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FIG. 2. Sensitivity of PG9 and PG16 neutralization to substitutions eliminating N-linked glycosylation sites. (A) JR-CSF PG9; (B) JR-CSF PG16; (C) JR-FL E168K PG9; (D) JR-FL E168K PG16.

lication (15, 20, 27, 55), whereas inhibitors of the later stages of N-linked glycan processing (e.g., deoxymannojirimycin [DMJ], kifunensine, and swainsonine) have no effect (11, 20, 41). We generated pseudovirus in the presence of kifunensine, swainsonine, and NB-DNJ to investigate the effect of the type of glycan present on sensitivity to PG9 and PG16 neutralization. Pseudovirus was made in the presence of kifunensine, a glycosidase inhibitor that prevents the trimming of Man9GlcNAc2 by the ER-mannosidase I enzyme in the ER, thus resulting in pseudovirus displaying only Man9GlcNAc2 at all utilized Nlinked glycosylation sites (Fig. 3). Indeed, Scanlan et al. have shown that recombinant production of gp120 in CHO cells in the presence of kifunensine results in all N-linked glycosylation sites being occupied with Man9GlcNAc2 (52) and fully homogeneous glycosylation. As a control for the effect of kifunensine on pseudoviral particles, 2G12 was used to show enhanced neutralization due to additional high-mannose binding sites (52), and b12 was used as a negative control as an increased level of high-mannose glycans does not affect b12 binding (Fig. 4A and B) (52). The kifunensine JR-CSF pseudovirus was no longer sensitive to neutralization by PG9 and PG16, indicating that the presence of extra high-mannose glycans on the HIV trimer prevented binding by PG9 and PG16 (Fig. 4C and D). This may be the result of a conformational change, probably in the V1/V2 and/or V3 loops resulting from the change in glycosylation pattern, a change of a glycan interacting directly with PG9 and PG16, or masking of the protein epitope due to the presence of the larger Man9GlcNAc2 gly-

can. The lack of competition of mannose with PG9 and PG16 for envelope binding argues against the second interpretation. Kifunensine had a similar effect on PG9 and PG16 binding to pseudovirus using JR-FL E168K (Fig. 5C and D), 92RW020 (Fig. 6A and B), and SF162 K160N (Fig. 6C and D) envelopes. Overall, the results, together with data suggesting that treatment of gp120DU422 with Endo H significantly decreased PG9 binding, suggest that changes in protein conformation or epitope accessibility rather than direct contact of glycans with the PG9 binding site are responsible for sensitivity of PG9 neutralization to glycans (56). Pseudovirus was also made in the presence of the glycosidase inhibitor swainsonine, which inhibits the enzyme Golgi␣-mannosidase II (Fig. 3) (10). Swainsonine prevents the formation of complex sugars by inhibiting the trimming of mannose residues from GlcNAcMan5GlcNAc2. Since we have shown that HIV-1 viral envelope consists mainly of oligomannose glycans (9), treatment with swainsonine should not cause changes to the glycosylation profile other than removal of trace amounts of complex glycans and/or alterations to the positioning of oligomannose glycans on the virus surface. As expected, neutralization of swainsonine-treated JR-CSF pseudovirus (swain-JR-CSF) and swain-JR-FL E168K by b12 and 2G12 was similar to that of wild-type viruses (Fig. 4A, 4B, 5A, and 5B). Neutralization of swain-JR-CSF by PG9 and PG16 was also similar to that of the wild-type virus (Fig. 4C and D). However, neutralization of swain-JR-FL E168K by PG9 and PG16 showed a neutralization profile similar to that of JR-CSF and

FIG. 3. N-linked glycosylation pathway to show the formation of high-mannose, complex, and hybrid glycans. Glycosidase inhibitors (red) are shown underneath the enzyme they inhibit. 10514

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FIG. 4. Neutralization activity of MAbs against JR-CSF pseudoviruses made in the presence of glycosidase inhibitors. (A) b12; (B) 2G12; (C) PG9; (D) PG16.

distinct from the shallow sloping curve that plateaued below 100% seen for wild-type JR-FL E168K (Fig. 5C and D). Neutralization of swain-92RW020 and swain-SF162 K160N by PG9 was similar to that for the wild-type viruses (Fig. 6A and C). PG16 was able to neutralize swain-SF162 K160N to ca. 90% compared to ca. 75% for the wild-type virus (Fig. 6B and D), and a slight increase in neutralization sensitivity of swain92RW020 by PG16 was observed. Overall, viruses made in the presence of swainsonine were similarly or somewhat more efficiently neutralized by PG9 and PG16. NB-DNJ treatment of HIV leads to a reduction in HIV infectivity (12, 13), with virus unable to undergo productive post-receptor binding rearrangement or fusion with the cell membrane of the target cell (12–14). This effect is suggested to arise from changes in gp120 V1 and V2 conformation (regions involved in the PG9 and PG16 epitopes) (13). NButyldeoxynojirimycin (NB-DNJ) inhibits ␣-glucosidase I and II in the ER, and removal of the glucose residues from Man9GlcNAc2 does not occur, resulting in pseudovirus displaying Glc1-3Man9GlcNAc2 glycans (Fig. 3) (12–14, 26). However, in HEK-293T cells there is an endomannosidase present that can cleave between the terminal mannose residues of the D1 arm while glucose residues are still attached (49), thus representing an alternative pathway to more processed glycans (Fig. 3). Therefore, virus made in the presence of NB-DNJ in HEK-293T cells will display Glc1-3Man7-9GlcNAc2 glycans in addition to the “normal” glycans found on the envelope protein. Here, we found that NB-DNJ-JR-CSF and NB-DNJJR-FL E168K isolates were both insensitive to neutralization by 2G12 (Fig. 4B and 5B), as expected, since the Man␣1,2Man

linkages of the D1 arm of Man9GlcNAc2 are either capped with glucose residues preventing 2G12 binding or cleaved by the endomannosidase (4, 49, 51). However, neutralization by b12 remained unchanged after NB-DNJ treatment (Fig. 4A and B). The neutralizing activity of PG9 and PG16 against NB-DNJ-JR-CSF was similar to the untreated virus, whereas NB-DNJ-JR-FL E168K was much more sensitive to neutralization after 2 mM NB-DNJ treatment (Fig. 4C, 4D, 5C, and 5D). The infectivity of 92RW020 and SF162 K160N viruses made in the presence of NB-DNJ was insufficient to gain accurate neutralization data. Virus was also made in the presence of a combination of kifunensine and NB-DNJ. The resulting virus will display both Glc1-3Man9GlcNAc2 and Man8GlcNAc2 glycans. Both PG9 and PG16 were unable to neutralize either JR-CSF or JR-FL E168K prepared with these two inhibitors, as seen with virus prepared with kifunensine alone. The infectivity of these NB-DNJ-treated viruses was somewhat lower than that of the wild-type viruses, but infectivity was still significant, and no effects on neutralization by b12 were observed. In another strategy, pseudoviruses were produced in the mutant GnT1-deficient HEK-293S cell line (GnT1⫺/⫺) (47). These cells are deficient in N-acetylglucosaminyltransferase I, and addition of GlcNAc residues to the Man5GlcNAc2 structure in the Golgi does not occur (Fig. 3). We have shown that viruses grown in these cells display an increased amount of Man5GlcNAc and a decreased amount of Man6-9GlcNAc glycans (9) compared to viruses produced in 293T cells. For JRCSF pseudovirus, decreased neutralization sensitivity was observed for both PG9 and PG16 (Fig. 4C and D). For 293SJR-FL-E168K and 293S-SF162-K160N pseudoviruses, the

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FIG. 5. Neutralization activity of MAbs against JR-FL E168K pseudoviruses made in the presence of glycosidase inhibitors. (A) b12; (B) 2G12; (C) PG9; (D) PG16.

shallow sloping neutralization profile and the ⬍100% plateau of PG16 were not observed, and a normal sigmoidal curve was seen (Fig. 5D, 6C, and 6D). For 293S-92RW020 virus the low neutralization plateau was still observed for PG16 (Fig. 6A and B). Neutralization by b12 and 2G12 remained unchanged as before with the swain-pseudoviruses (Fig. 4A, 4B, 5A, and 5B). Therefore, the glycan profile of virus produced in 293S cells results in enhanced sensitivity to neutralization by PG9 and PG16 in the majority of viruses that showed aberrant neutralization profiles but also a reduction in neutralization sensitivity for some isolates examined. Effect of swainsonine treatment on trimer affinity of PG9 and PG16. We next investigated whether the affinity of PG9 and PG16 for the cleaved HIV trimer on JR-FL E168K-transfected 293T cells differed when the cells were transfected in the presence or absence of the glycosidase inhibitor swainsonine. The affinity of these antibodies for these trimers differed only slightly (Fig. 7). However, the plateau of binding for both PG9 and PG16 for the wild-type cells was lower than that for the swainsonine-treated cells (11 and 16% reductions, respectively). This value is similar to the difference in neutralization plateaus of PG9 and PG16 (9 and 16% reduction, respectively) against the corresponding pseudoviruses. The b12 control showed no difference in binding plateau. This suggests that a fraction of different glycosylated trimer molecules are unable to bind to PG9 and PG16. Mixed trimer experiment: We investigated the neutralization activity of PG9 and PG16 on viruses displaying trimers

that contained both a monomer that results in the shallow sloping curve and a monomer that results in the normal sigmoidal neutralization curve. We have previously shown that although PG9 and PG16 are trimer specific, these antibodies do not cross-link gp120 molecules within the same trimer (56). This suggests that even if one gp120 molecule displays the “optimal” glycans that give rise to the PG9 and PG16 epitopes, then this could be sufficient for 100% neutralization (56). We hypothesized that the conformation of one gp120 molecule in a trimer may be able to influence the conformation of the two other gp120 molecules. Therefore, if one gp120 molecule displayed glycans that gave a conformation unrecognizable by PG9 and PG16 this molecule may influence the whole trimer, preventing PG9 and PG16 neutralization. Since glycosidase inhibitors can only be used to change the whole virus population, we chose to use JR-FL E168K as the “suboptimally” glycosylated monomer (displaying the shallow neutralization profile) and JR-FL E168K N189A as the “optimally” glycosylated monomer (displaying the sigmoidal neutralization profile). Pseudovirus was therefore made by cotransfecting with both JR-FL E168K (displaying the shallow sloping neutralization curve) and JR-FL E168K N189A (showing the normal sigmoidal curve) at various ratios to mimic some of the natural glycan heterogeneity found in a solution of pseudovirus and to test this hypothesis (53, 59). The neutralization profiles of these chimeric viruses by PG9, PG16, and 2G12 are shown in Fig. 8. The percentage neutralization at 10 ␮g of antibody/ml was plotted against the percentage of variant envelope (Fig.

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FIG. 6. Neutralization activity of MAbs against 92RW020 and SF162 K160N pseudoviruses when made in the presence of glycosidase inhibitors. (A) 92RW020 PG9; (B) 92RW020 PG16; (C) SF162 K160N PG9; (D) SF162 K160N PG16.

8D). A binomial distribution of trimers was assumed and a one minimal “optimal” monomer requirement was fitted better than a two minimal “optimal” monomer requirement (53, 59). The data suggest that only one “optimal” subunit in a trimer is required to achieve full neutralization (Fig. 8). DISCUSSION Previous data showed that the neutralization activities of the broadly neutralizing antibodies PG9 and PG16 were dependent on the N-linked glycosylation at site N160 and that removal of high-mannose glycans from gp120DU422 using Endo H significantly reduced PG9 binding (56). This suggested that the PG9 and PG16 epitopes were dependent on glycosylation of the envelope trimer. It was also observed that the neutral-

ization profile of these antibodies can, in some cases, show a shallow sloping curve that plateaus at less than 100% (56). This led to the hypothesis that glycan heterogeneity and the glycan profile of the trimer could affect the ability of PG9 and PG16 to neutralize pseudovirus. We set out to manipulate the glycosylation of the trimer in order to investigate the glycan dependency of these antibodies. A glycoprotein can differ in the type of glycosylation displayed at a given N-linked glycosylation site. Therefore, a population of glycoproteins consists of different species (glycoforms) in which the protein backbone is identical but the glycans differ. Since glycosylation of a protein is known to have a significant effect on protein conformation (50), these glycoforms may have different conformations. We have recently

FIG. 7. Cell surface binding of MAbs to swainsonine-treated HIV JR-FL E168K Env-transfected cells compared to untreated HIV JR-FL E168K Env cells. (A) PG9; (B) PG16; (C) b12.

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FIG. 8. Neutralization of glycan mixed trimer viruses by PG9, PG16, and b12. Cells were transfected with various proportions of JR-FL E168K and JR-FL E168K N189A. The percentage refers to the amount of JR-FL E168K N189A plasmid used. (A) PG9; (B) PG16; (C) 2G12. (D) The percent neutralization at an antibody concentration of 10 ␮g/ml was plotted against the ratio of “suboptimal” envelope (i.e., ratio of JR-FL E186K). The data fit best to the equation N ⫽ Nplateau ⫹ [(1 ⫺ X)3 ⫹ 3X(1 ⫺ X)2 ⫹ 3(1 ⫺ X)X2](Nmax ⫺ Nplateau), where N is the neutralization and X is the ratio of suboptimal substitution (i.e., only one “optimal” monomer is required per trimer to reach maximum neutralization) (53, 59).

shown that the glycosylation of HIV-1 envelope is dominated by Man5-9GlcNAc2 glycans (9). Although this suggests much less glycan heterogeneity than previously thought (7, 18, 19, 61), multiple HIV-1 trimer glycoforms do exist. It appears that PG9 and PG16 have nonlinear, conformational epitopes that are strongly dependent on glycosylation (56), so it is possible that, within a population of trimer glycoforms, there will be molecules that PG9 and PG16 either cannot bind or bind with lowered affinity. Many studies in the literature that investigate the importance and effects of glycosylation on HIV-1 focus on the presence or absence of a glycosylation site (5, 28, 36, 42). Glycans are known to shield peptide epitopes (48) and are poorly immunogenic, so it is not surprising that removal of glycosylation sites by site-directed mutagenesis leads to a more sensitive virus and a more immunogenic protein (28, 34, 38, 45). It should be noted that removal of an N-linked glycosylation site can have more profound effects than just the loss of that particular glycan. First, the loss of a glycan may affect the conformation of the protein. Second, the loss of a glycan from a clustered arrangement of glycans may allow an increased accessibility of the glycan processing enzymes to the cluster, resulting in a change in the glycan profile of the protein. This in turn could affect the conformation of the protein in an area distant from the lost glycan. For neutralization of HIV by PG9 and PG16, it seems that removal of N-linked glycosylation sites generally leads to a reduction in sensitivity to the antibodies (although some in-

creased sensitivity was also observed for neutralization of the JR-FL E168K variants by PG16). Reduced neutralization sensitivity is particularly apparent for loss of glycosylation sites in the V1, V2, and V3 loops and is most apparent for PG16 (Fig. 2). The position of the N-linked glycosylation site removed and the magnitude of the effect appear to be isolate dependent (see below for discussion). The binding of PG9 and PG16 to Envtransfected cells and PG9 to recombinant gp120 was not competed by monosaccharides, suggesting that, for the majority of variants, antibody sensitivity to glycosylation is more likely associated with the effect of glycans on protein conformation than on direct binding of antibody to glycans. It appears that the N160 glycan has the most significant role in maintaining the PG9 and PG16 conformational epitopes since this is the only substitution to universally eliminate sensitivity to neutralization (Fig. 2). We have previously shown that PG16 is more sensitive to substitutions in the V3 region than PG9 (56), and we have now shown that PG16 is also more sensitive to the removal of N-linked glycosylation sites. N-linked glycosylation sites in the V1/V2 loops can mask the V3 loop, suggesting some direct or indirect interaction between these loops (5, 36). Since the effect of removing N-linked glycosylation sites in the V1/V2 loops leads to decreased PG16 neutralization activity, it seems likely that these glycans may not be masking the PG16 epitope but rather helping to maintain the correct conformation of the V3 loop to allow optimal recognition by PG16. It was hypothesized that the glycan composition of the envelope could be affecting the neutralization activity of PG9 and

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GLYCAN DEPENDENCY OF PG9 AND PG16

TABLE 1. Summary of changes in neutralization profile of PG9 and PG16 with viruses prepared with glycosidase inhibitors compared to wild-type virus Neutralization profilea Antibody and HIV-1 isolate

Kifunensine Swainsonine NB-DNJ 293S

NB-DNJ, kifunensine

PG9 JR-CSF JR-FL E168K SF162 K160N 92RW020

0 0 0 0

⫽ ⫽* ⫽* ⫽

⫽ ⫽* ND ND

– ⫽* – ⫽

0 0 0 0

PG16 JR-CSF JR-FL E168K SF162 K160N 92RW020

0 0 0 0

⫽ –* ⫹* ⫹*

⫽ ⫽* ND ND

– –* –* –

0 0 0 0

a Score: 0, no neutralization; ⫽, no change in IC50; ⫹, increase in neutralization sensitivity; –, decrease in neutralization sensitivity; ⴱ, change in shape of neutralization profile or change in neutralization plateau; ND, not determined.

PG16. To test this hypothesis, we used inhibitors of the glycanprocessing enzymes (kifunensine, swainsonine, and NB-DNJ) (12–15, 20, 27, 41, 55) and also an N-acetylglucosaminyltransferase I (47)-deficient cell line (293S) to control glycan composition of the HIV-1 trimer. Glycan composition was shown to have a significant impact on the neutralization profile of PG9 and PG16 (Fig. 4, 5, and 6) and the results are summarized in Table 1. The presence of untrimmed high-mannose glycans (Man9GlcNAc2, Man8GlcNAc2, or Glc1-3Man9GlcNAc2) at sites additional to the 2G12 binding site (by expression in the presence of kifunensine or both NB-DNJ and kifunensine) resulted in the loss of neutralization activity for all isolates studied (Table 1). This consistent trend suggests that increased steric bulk of these particular glycans may occlude key PG9 and PG16 binding residues and/or alter the conformational epitopes of these antibodies. The universal and unique effect of kifunensine treatment on the sensitivity of PG9 and PG16 neutralization has lead to a diagnostic tool for identifying PG9 and PG16-like specificity in the serum of patients displaying broad neutralization (57). Generation of virus in the presence of NB-DNJ alone favors the binding of PG9 and PG16. It has previously been shown that generation of virus in the presence of NB-DNJ affects the conformation of the V1 and V2 loops, and it appears that the conformation of these loops in NBDNJ-JR-CSF and NB-DNJ-JR-FL E168K viruses favorably affects the conformation of the epitopes of PG9 and PG16. When the virus was prepared in GnT1-deficient 293S cells, which we have previously shown to display increased Man5GlcNAc2 glycans and decreased Man6-9GlcNAc2 glycans compared to those made in 293T cells (9), a decrease in neutralization sensitivity of JR-CSF was observed for both PG9 and PG16 neutralization. For the viruses displaying a neutralization plateau of ⬍ 100%, the change in glycan profile associated with growth in 293S cells produced sigmoidal neutralization profiles that reached almost 100% in most cases. An increased number of Man5GlcNAc2 glycans therefore favors binding to PG9 and PG16 to the trimer and neutralization for most isolates. This result, when combined with the kifunensine

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studies, suggests that there might be a proportion of viruses with Man8-9GlcNAc2 at a crucial N-linked glycosylation site that prevents trimer binding to PG9 and PG16 and, as there is a reduced number of these glycans on the virus made in 293S cells, binding of PG9 and PG16 is enhanced. An analysis of the glycans present at each N-linked glycosylation site would be required to understand this more fully but is beyond the scope of the present study. We also observed that when most viruses displaying a neutralization plateau of ⬍100% were prepared in the presence of swainsonine, normal sigmoidal neutralization profiles were generally produced (Table 1). Cell surface binding of PG9 and PG16 to HIV-transfected cells in the presence or absence of swainsonine revealed that there was no significant difference in affinity for trimers, but there was a higher binding plateau for the swainsonine-treated trimers (similar to the neutralization plateau, Fig. 7), further suggesting, for some isolates, that a portion of wild-type envelopes are not recognized by PG9 and PG16. The effect of either loss of a N-linked glycan or change in virus glycosylation pattern was found to be somewhat isolate and antibody dependent. In particular, we observed considerable variation between the PG9 and PG16 neutralization sensitivities of different HIV-1 isolates when generated in GnT1deficient 293S cells, in the presence of swainsonine or as N-linked glycosylation site knockouts (Table 1). The number of N-linked glycosylation sites, the occupancy of these sites, and both the length and sequence of the variable loops can vary significantly between isolates. This may subsequently impact the interaction of the V1, V2, and V3 loops such that when a glycan is removed or altered differing effects on the PG9 and PG16 epitopes occur depending on the HIV-1 isolate. Overall, it appears natural variation in the glycosylation at each N-linked glycosylation site results in a proportion of some isolates having glycoforms for which the optimal conformational epitope of PG9 and PG16 are not displayed. In order to dissect this more fully, an analysis of glycosylation at each position of the virus envelope would need to be determined and correlated with the effect of the removal of the glycan and change to neutralization sensitivity. Inefficient neutralization is observed with only 8% of the isolates neutralized by PG9 but 35% of the isolates neutralized by PG16 (56) and should be considered when carrying out passive protection studies in macaques, particularly with PG16. Vaccination should aim to elicit a polyclonal antibody response to the PG9/PG16 epitopes that should contain a spectrum of individual neutralizing specificities that are differentially sensitive to glycan heterogeneity providing coverage of most if not all isolate glycoforms. We have previously shown that although PG9 and PG16 preferentially bind trimers, they do not cross-link monomer units within the same trimer (56). A trimer may consist of different glycoforms of the monomer due to the heterogeneity of glycosylation (as discussed above). We have shown by creating viruses displaying mixed trimers consisting of “optimally” glycosylated monomers and “suboptimally” glycosylated monomers that only one “optimally” glycosylated monomer is required for complete neutralization (plateau at 100%) (Fig. 8). This suggests that the conformation of monomers within a trimer does not influence neighboring monomers but that, for some isolates, there are significant amount of trimers with all three monomers displaying the “suboptimal” glycosylation.

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DOORES AND BURTON

In summary, the results presented here show that the epitopes of PG9 and PG16 are dependent on the presence of N-linked glycosylation sites at certain positions in the V1, V2, and V3 loops, as well as glycan profile. This is particularly apparent for PG16 that shows decreased neutralization potency if one of several sites is substituted. We show that altering the glycosylation pattern on HIV-1 pseudoviruses using glycosidase inhibitors or a mutant cell line (GnT1⫺/⫺) has isolate-dependent effects on the profile of neutralization by PG9 and PG16. For viruses displaying an aberrant neutralization profile, alterations to the glycan profile leads to a normal sigmoidal neutralization profile in several, but not all, cases. Altered glycosylation profiles, with the exception of that associated with kifunensine treatment that universally results in decreased PG9 and PG16 neutralization sensitivity, result in increased, unaffected, or decreased neutralization sensitivity in an isolate-dependent manner. Viruses prepared in the presence of kifunensine are universally and uniquely insensitive to neutralization by PG9 and PG16, and this can be used to help identify the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. ACKNOWLEDGMENTS This study was funded by the International AIDS Vaccine Initiative through the Neutralizing Antibody Consortium, National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant AI33292 [D.R.B.]), and the Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard. We thank Laura Walker, Chris Scanlan, and Michael Huber for helpful discussions and Christina Corbaci for assistance with graphics. REFERENCES 1. Ben-Dor, S., N. Esterman, E. Rubin, and N. Sharon. 2004. Biases and complex patterns in the residues flanking protein N glycosylation sites. Glycobiology 14:95–101. 2. Burton, D. R. 2002. Antibodies, viruses, and vaccines. Nat. Rev. Immunol. 2:706–713. 3. 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. 4. 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. 5. Cole, K. S., J. D. Steckbeck, J. L. Rowles, R. C. Desrosiers, and R. C. Montelaro. 2004. Removal of N-linked glycosylation sites in the V1 region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3. J. Virol. 78:1525–1539. 6. Crispin, M. D., G. E. Ritchie, A. J. Critchley, B. P. Morgan, I. A. Wilson, R. A. Dwek, R. B. Sim, and P. M. Rudd. 2004. Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure. FEBS Lett. 566:270–274. 7. 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. 8. de Witte, L., A. Nabatov, and T. B. Geijtenbeek. 2008. Distinct roles for DC-SIGN⫹-dendritic cells and Langerhans cells in HIV-1 transmission. Trends Mol. Med. 14:12–19. 9. Doores, K. J., C. Bonomelli, D. J. Harvey, S. Vasiljevic, R. A. Dwek, D. R. Burton, M. Crispin, and C. N. Scanlan. 2010. Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc. Natl. Acad. Sci. U. S. A. 107:13800–13805. 10. Elbein, A. D., R. Solf, P. R. Dorling, and K. Vosbeck. 1981. Swainsonine: an inhibitor of glycoprotein processing. Proc. Natl. Acad. Sci. U. S. A. 78:7393– 7397. 11. Elbein, A. D., J. E. Tropea, M. Mitchell, and G. P. Kaushal. 1990. Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I. J. Biol. Chem. 265:15599–15605. 12. Fischer, P. B., M. Collin, G. B. Karlsson, W. James, T. D. Butters, S. J. Davis, S. Gordon, R. A. Dwek, and F. M. Platt. 1995. The alpha-glucosidase

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