Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Human immunodeficiency virus envelope (gp120) binding to DC-SIGN and primary dendritic cells is carbohydrate dependent but does not involve 2G12 or cyanovirin binding sites: implications for structural analyses of gp120-DC-SIGN binding

The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-beta-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding-and the full complement of complex glycans-significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.     

HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.     

Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.     

The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1-->2 mannose residues on the outer face of gp120

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N-->A substitution produced significant decreases in 2G12 binding affinity to gp120(JR-CSF). Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120(JR-CSF). Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Man(alpha)1-->2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Man(alpha)1-->2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Man(alpha)1-->2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.     

Restraining the conformation of HIV-1 gp120 by removing a flexible loop

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.     

Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate HIV infection in cis and in trans, is largely dependent on high-mannose-content moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute to optimal DC-SIGN binding. Soluble DC-SIGN was able to block 2G12 binding to gp120, but not vice versa, suggesting that DC-SIGN binds to a more flexible combination of N-glycans than 2G12. Consistent with this observation, HIV strain JRCSF gp120 prebound to 2G12 was 10-fold more sensitive to mannan competition than gp120 that was not prebound in a DC-SIGN cell surface binding assay. The analysis of multiple mutant forms of the 2G12 epitope revealed one triple glycosylation mutant form, termed 134mut (carrying N293Q, N382Q, and N388Q mutations), that exhibited a significant increase in sensitivity to both mannan competition and endoglycosidase H digestion compared to that of the 124mut form (carrying N293Q, N328Q, and N388Q mutations) and wild-type gp120 in a DC-SIGN binding assay. Importantly, no such differences were observed when binding to Galanthus nivalis was assessed. The 134mut form of gp120 also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and virus bearing 134mut exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site mutation in each construct, and both 124mut and 134mut viruses exhibited wild-type levels of infectivity when used in a direct infection assay. In summary, while DC-SIGN can bind to a flexible combination of N-glycans on gp120, its optimal binding site overlaps with specific N-glycans within the 2G12 epitope. Conformationally intact envelopes that are DC-SIGN binding deficient can be used to probe the in vivo biological functions of DC-SIGN.     

The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120

We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.     

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.     

Concanavalin A binding to HIV envelope protein is less sensitive to mutations in glycosylation sites than monoclonal antibody 2G12

Many mannose-binding proteins inhibit divergent strains of human immunodeficiency virus type 1 (HIV-1) in in vitro models of viral infectivity, suggesting that targeting mannose residues in vaccine applications might offset the strain restriction typically observed in antibody responses to HIV vaccine preparations. Concanavalin A (ConA) behaves like neutralizing antibodies that do not interfere with CD4 binding of gp120 but rather with later events in virus entry. The design of mannose-based vaccines, therefore, depends on understanding the mode of binding of ConA to the envelope protein in comparison with other mannose-binding proteins. Here, we further compare the binding affinity and fine specificity of ConA for the envelope protein to that of the human antibody 2G12. The 2G12 antibody is of unusual structure recognizing a cluster of 12 linked mannose residues associated with Man9GlcNAc2. Molecular structure comparison for Man9GlcNAc2 recognition by ConA and 2G12 indicates that 2G12 has a more restricted specificity to high mannose glycans of gp120 which correlates with kinetic analysis assessed by surface plasmon resonance (SPR) and ConA inhibits 2G12 binding to gp120 but 2G12 does not inhibit ConA binding to gp120. ConA binding to Env proteins from four different HIV strains proves significantly less sensitive to mutations in the glycosylation sites than 2G12 binding to the proteins. Thus, antibodies directed toward mannose epitopes reactive with ConA may prove to be more effective in the long run to thwart HIV infection and transmission.     

Solution structure of the monovalent lectin microvirin in complex with Man(alpha)(1-2)Man provides a basis for anti-HIV activity with low toxicity

Lectins that bind surface envelope glycoprotein gp120 of HIV with high avidity can potently inhibit viral entry. Yet properties such as multivalency that facilitate strong interactions can also cause nonspecific binding and toxicity. The cyanobacterial lectin microvirin (MVN) is unusual as it potently inhibits HIV-1 with negligible toxicity compared with cyanovirin-N (CVN), its well studied antiviral homolog. To understand the structural and mechanistic basis for these differences, we solved the solution structure of MVN free and in complex with its ligand Manα(1-2)Man, and we compared specificity and time windows of inhibition with CVN and Manα(1-2)Man-specific mAb 2G12. We show by NMR and analytical ultracentrifugation that MVN is monomeric in solution, and we demonstrate by NMR that Manα(1-2)Man-terminating carbohydrates interact with a single carbohydrate-binding site. Synchronized infectivity assays show that 2G12, MVN, and CVN inhibit entry with distinct kinetics. Despite shared specificity for Manα(1-2)Man termini, combinations of the inhibitors are synergistic suggesting they recognize discrete glycans and/or dynamic glycan conformations on gp120. Entry assays employing amphotropic viruses show that MVN is inactive, whereas CVN potently inhibits both. In addition to demonstrating that HIV-1 can be inhibited through monovalent interactions, given the similarity of the carbohydrate-binding site common to MVN and CVN, these data suggest that gp120 behaves as a clustered glycan epitope and that multivalent-protein interactions achievable with CVN but not MVN are required for inhibition of some viruses.     

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.     

N-linked glycan modifications in gp120 of human immunodeficiency virus type 1 subtype C render partial sensitivity to 2G12 antibody neutralization

The monoclonal antibody (MAb) 2G12 recognizes a cluster of high-mannose oligosaccharides on the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 and is one of a select group of MAbs with broad neutralizing activity. However, subtype C viruses are generally resistant to 2G12 neutralization. This has been attributed to the absence of a glycosylation site at position 295 in most subtype C gp120s, which instead is typically occupied by a Val residue. Here we show that N-linked glycans in addition to the one at position 295 are important in the formation of the 2G12 epitope in subtype C gp120. Introduction of the glycosylation site at position 295 into three subtype C molecular clones, Du151.2, COT9.6, and COT6.15, did increase 2G12 binding to all three mutagenized gp120s, but at various levels. The COT9-V295N mutant showed the strongest 2G12 binding and was the only mutant to become sensitive to 2G12 neutralization, although very high antibody concentrations were required. Introduction of a glycosylation site at position 448 into mutant COT6-V295N, which occurs naturally in COT9, resulted in a virus that was partially sensitive to 2G12. Interestingly, a glycosylation site at position 442, which is common among subtype C viruses, also contributed to the 2G12 epitope. The addition of this glycan increased virus neutralization sensitivity to 2G12, whereas its deletion conferred resistance. Collectively, our results indicate that the 2G12 binding site cannot readily be reconstituted on the envelopes of subtype C viruses, suggesting structural differences from other HIV subtypes in which the 2G12 epitope is naturally expressed.     

Gold nanoparticles coated with oligomannosides of HIV-1 glycoprotein gp120 mimic the carbohydrate epitope of antibody 2G12

After three decades of research, an effective vaccine against the pandemic AIDS caused by human immunodeficiency virus (HIV) is not still available, and a deeper understanding of HIV immunology, as well as new chemical tools that may contribute to improve the currently available arsenal against the virus, is highly wanted. Among the few broadly neutralizing human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies, 2G12 is the only carbohydrate-directed one. 2G12 recognizes a cluster of high-mannose glycans on the viral envelope glycoprotein gp120. This type of glycan has thus been envisaged as a target to develop an HIV vaccine that is capable of eliciting 2G12-like antibodies. Herein we show that gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides [manno-gold glyconanoparticles (GNPs)], which are present in gp120, are able to bind 2G12 with high affinity and to interfere with 2G12/gp120 binding, as determined by surface plasmon resonance and saturation transfer difference NMR spectroscopy. Cellular neutralization assays demonstrated that GNPs coated with a linear tetramannoside could block the 2G12-mediated neutralization of a replication-competent virus under conditions that resemble the ones in which normal serum prevents infection of the target cell. Dispersibility in water and physiological media, absence of cytotoxicity, and the possibility of inserting more than one component into the same nanoparticle make manno-GNPs versatile, polyvalent, and multifunctional systems that may aid efforts to develop new multifaceted strategies against HIV.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

Proline Is Not Uniquely Capable of Providing the Pivot Point for Domain Swapping in 2G12, a Broadly Neutralizing Antibody against HIV-1

The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the “silent” face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.     

Envelope glycoprotein determinants of neutralization resistance in a simian-human immunodeficiency virus (SHIV-HXBc2P 3.2) derived by passage in monkeys

The simian-human immunodeficiency virus SHIV-HXBc2 contains the envelope glycoproteins of a laboratory-adapted, neutralization-sensitive human immunodeficiency virus type 1 variant, HXBc2. Serial in vivo passage of the nonpathogenic SHIV-HXBc2 generated SHIV KU-1, which causes rapid CD4(+) T-cell depletion and an AIDS-like illness in monkeys. A molecularly cloned pathogenic SHIV, SHIV-HXBc2P 3.2, was derived from the SHIV KU-1 isolate and differs from the parental SHIV-HXBc2 by only 12 envelope glycoprotein amino acid residues. Relative to SHIV-HXBc2, SHIV-HXBc2P 3.2 was resistant to neutralization by all of the antibodies tested with the exception of the 2G12 antibody. The sequence changes responsible for neutralization resistance were located in variable regions of the gp120 exterior envelope glycoprotein and in the gp41 transmembrane envelope glycoprotein. The 2G12 antibody, which neutralized SHIV-HXBc2 and SHIV-HXBc2P 3.2 equally, bound the HXBc2 and HXBc2P 3.2 envelope glycoproteins on the cell surface comparably. The ability of the other tested antibodies to achieve saturation was less for the HXBc2P 3.2 envelope glycoproteins than for the HXBc2 envelope glycoproteins, even though the affinity of the antibodies for the two envelope glycoproteins was similar. Thus, a highly neutralization-sensitive SHIV, by modifying both gp120 and gp41 glycoproteins, apparently achieves a neutralization-resistant state by decreasing the saturability of its envelope glycoproteins by antibodies.     

Analysis of the site in CD4 that binds to the HIV envelope glycoprotein.

The first step in infection of human mononuclear cells with HIV involves the high affinity binding of the viral envelope glycoprotein, gp120, to the cell-surface receptor, CD4. To gain a better understanding of the molecular basis of this interaction, we have analyzed the ability of gp120 to bind to a panel of 40 mutant CD4 proteins containing single or double amino acid substitutions. In addition, the binding of several anti-CD4 mAb to the mutant CD4 proteins was measured. These mAb were chosen on the basis of the previous demonstration that they bind to epitopes in CD4 adjacent to the gp120-binding site. This analysis permits discrimination between mutations that probably cause localized conformational changes and those that alter residues likely to make direct contact with gp120 and with the mAb. Our results indicate that gp120 from two different strains of HIV binds to a larger region of the CD4 protein than previously described. The data has also been used to map the epitopes of mAb previously identified as anti-idiotype vaccine candidates. The results have important implications for the development of CD4-based therapies for AIDS.