Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC₅₀) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.Immunoglobulin molecules have multiple binding paratopes for antigens; for example, those for IgG1 are bivalent and those for IgM are dodecavalent. It is obvious that multivalent binding is required for the distinct mechanism of neutralization by cross-linking multiple virions to form virus aggregates (reviewed in references 7 and 67). Despite the potential of antibodies for multivalent binding, structural evidence indicates that neutralizing antibodies often bind to an individual virion in a monovalent fashion (19, 20, 27, 29, 50, 53; reviewed in references 12 and 22). Bivalent binding of an antibody to a virion has been documented with clear structural evidence in only one case, in which monoclonal antibodies (MAbs) 17-IA and 8F5 bind to virions of human rhinovirus 14 (HRV14) and HRV2 (19, 43). Even in this unique case, binding bivalency appears to contribute to the neutralization potency of 17-IA but not to that of 8F5 (19, 42, 43). Moreover, these MAbs bind to two hydrophobic canyon structures formed by viral proteins VP1 and VP2 and not to antigenic epitopes within individual viral capsid protomers; thus, this case may represent an exception to the common form of antibody/antigen interactions in which the antibodies bind to individual antigens. Therefore, it is not clear what role antibody-binding multivalency plays in antibody-mediated neutralization of viruses at the level of interaction between antibody molecules and individual virions.The binding affinity of an antibody to its target is defined by intrinsic affinity and avidity (reviewed in reference 16). Intrinsic affinity is the force of monovalent binding between an antibody paratope and an antigenic epitope, often measured by binding a Fab fragment to an antigen. Avidity is the additive or synergistic force of engaging multiple antibody paratope/antigen epitope pairs between one antibody and one antigen. In other words, avidity is a functional consequence of antibody-binding multivalency. The effect of avidity on affinity is readily demonstrated in biochemical reactions such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), in which high-density antigenic sites are available without distinct spatial restrictions. It is commonly assumed that both affinity and avidity have functional consequences in antibody-mediated neutralization of viruses (reviewed in references 7 and 67). At the level of individual virions, the contribution of antibody-binding avidity to neutralization efficiency is often based on two types of experiments. In one, results from a side-by-side comparison between an antibody and its Fab fragment are often reported as evidence supporting a role of antibody-binding multivalency in virus neutralization. However, the interpretation of this type of experiment is complicated by the size difference between an antibody and a Fab fragment, since steric hindrance is a major mechanism of neutralization (reviewed in references 6 and 23). In a second type of experiment, a correlation between neutralization efficiency and the ability of the antibody/virus complex to resist chemical stress without dissociation in the presence of a high concentration of salt in solution is interpreted to support a contributing effect from antibody-binding avidity to neutralization efficiency (2, 21, 36, 49, 51). Data from this type of experiment are limited mostly to measuring binding affinity that is below the affinity required for virus neutralization. Furthermore, these studies often do not distinguish between avidity effects caused by an antibody binding to two (or more) epitopes on one antigen or to multiple epitopes from different molecules on the virion. Therefore, like the situation with antibody-binding multivalency, it remains unclear whether binding avidity contributes to antibody-mediated neutralization of viruses at the level of individual virions.The envelope glycoproteins (Envs) of human immunodeficiency virus type 1 (HIV-1) exist on the virion or cell surface as trimers of gp120 and gp41 heterodimers (13, 30, 62, 65). High-resolution structural information for a native HIV-1 Env trimer is critically important for understanding the function of HIV-1 Envs as well as for guiding the development of an effective immunogen to elicit broad and potent neutralizing antibody responses. X-ray crystal structures of the gp41 ectodomain fragments in the postfusion conformation have been resolved; however, a high-resolution structure of gp41 in the prefusion conformation is still unavailable and likely will be more informative for understanding the function of HIV-1 Env trimers (9, 47, 52). Two X-ray crystal structures of the gp120 core in both the CD4-liganded and unliganded conformations have been solved, but the biological meanings of these structures, especially how they are related to the native, functional Env trimer, are still being debated (10, 26). Several low-resolution structures of the Env trimers from HIV-1 or the closely related simian immunodeficiency virus (SIV) have been determined using cryoelectron microscopy (cryo-EM) tomography (4, 30, 62, 64, 65, 66). The predicted structures for the Env trimer are in general quite different between the two studies, and the difference is particularly dramatic around the gp41 membrane-proximal external region (MPER). A high-resolution structure of the native HIV-1 Env trimer is needed to resolve these differences. In the meantime, a distinctive standard needs to be developed for evaluating the biological relevance of structural information of an HIV-1 Env trimer.Our previous studies of the stoichiometry of antibody-mediated neutralization of HIV-1 Env indicated that MAbs b12, 2G12, and 2F5 neutralize by a stoichiometry designated T=1, i.e., one antibody binds to and neutralizes one HIV-1 Env trimer (57). Furthermore, when an artificial epitope (FLAG) was inserted in the V4 region of HIV-1 gp120, an epitope-specific anti-FLAG MAb achieved neutralization by the mechanism of steric hindrance (37, 61). Using the well-defined 2F5 neutralizing epitope as a model system (35, 39, 45), we constructed HIV-1 Env proteins carrying one 2F5 epitope in the gp120 V4 region and another 2F5 epitope in the gp41 MPER. Here, we investigated whether binding bivalency leads to enhancement in neutralization efficiency. By studying the detailed requirement for binding bivalency, we also probed the structure of the native, functional HIV-1 Env trimer, aiming to establish a standard that can be employed to evaluate the biological relevance of structural information on the HIV-1 Env trimer.     

Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.     

Role of the Membrane-Proximal Domain in the Initial Stages of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein-Mediated Membrane Fusion

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315–323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

The human immunodeficiency virus type 1 (HIV-1) gp120-gp41 fusion machine consists of an assembly of viral envelope glycoprotein oligomers which forms a molecular scaffold responsible for bringing the viral membrane close to the target cell membrane and creating the architecture that enables lipid bilayers to merge (7). The fusion reaction undergoes multiple steps before the final event occurs which allows delivery of the nucleocapsid into the cell. In the case of influenza virus hemagglutinin (HA), we have dissected these steps kinetically and analyzed the molecular features of the kinetic intermediates (1). In order to examine the modus operandi of the fusion machine, mutations in various domains of viral envelope glycoproteins have been examined for their effect on the outcome of the fusion cascade. For instance, replacement of the membrane-spanning domain of influenza virus HA with a glycosylphosphatidylinositol anchor results in a very stable hemifusion intermediate (6). Moreover, single-site mutations in the fusion peptide of HA significantly affect fusion pore dilation (9). Recently, cytoplasmic tail acylation mutants of influenza virus HA were identified which induce transfer of lipids and small aqueous molecules but do not induce syncytium formation (4a).High-resolution crystallographic determinations (4, 10, 11) of gp41 fragments from HIV-1 have revealed a bent-in-half, antiparallel, heterotrimeric coiled-coil structure. This is made up of a triple-stranded coiled coil of α-helices from the leucine zipper-like 4-3 repeat domain in gp41 close to the N-terminal fusion peptide termed HR1 (8) flanked by α-helices from the domain in gp41 close to the C-terminal membrane anchor termed HR2 (8). Comparison with the crystal structure of the influenza virus HA2 subunits in a low-pH-induced conformation (2) reveals common structural motifs which provide growing support for the “spring-loaded” type of mechanistic models (3). In this scenario, activation of the fusion protein results in release of the fusion peptide and extension of the central coiled-coil structure. The new positioning of the fusion peptides at the tip of the stalk provides for easy contact with the target cell membrane. A small group of proximal fusion proteins which are simultaneously inserted into both the viral and target membranes would constitute a potential fusion site. A concerted collapse of this protein complex, actuated by the bending in half of the stalks at a central hinge region, would presumably position the C-terminal transmembrane anchors and N-terminal fusion peptides on top of each other in the center, bring the two membranes into contact, and thus allow formation of the fusion pore (7). In this study, we examined the effects on the various stages of the fusion reaction of mutations in the region between HR2 and the transmembrane (TM) anchor (Fig. ​(Fig.1)1) described in detail by Salzwedel et al. (8a). Open in a separate windowFIG. 1Amino acid sequence and mutations in gp41. FP is the predicted fusion peptide region, and HR1 and HR2 (8) represent, respectively, the N-terminal and C-terminal α-helices of the triple-stranded coiled coil (4, 11). Mutations in the region between HR2 and the TM anchor include deletions of amino acids 665 to 682 and 678 to 682, insertion of a FLAG sequence (YKDDDD), insertion of a DAF sequence (PNKGSGTTS), scrambling of the underlined sequence to SC7 (INNWNFT), and replacement of the five tryptophans with alanines [W(1-5)A]. Peptide C34 represents HR2 amino acids 628 to 663, and peptide DP178 represents amino acids 638 to 673.Mutagenesis of HIV-1 env, construction of plasmids, cell surface expression, CD4 binding, and cell fusion were performed as previously described (8a). The simian virus 40-based env expression plasmids (1 μg of DNA) were transfected into COS-1 cells in 35-mm-diameter plates by using DEAE-dextran (1 mg/ml). At 14 h posttransfection, the cells were replated, and starting at 36 to 48 h posttransfection, they were incubated with 20 μM CMAC (7-amino-4-chloromethylcoumarin) in Dulbecco modified Eagle medium overnight at 37°C. All constructs expressed similar amounts of envelope glycoprotein on the cell surface (8a). The transfected cells were then washed and incubated in fresh medium for 2 h at 37°C before addition of HeLa-CD4 cells which were labeled in the membrane with octadecyl indocarbocyanine (DiI) and in the cytosol with calcein as previously described (7). The method used to detect cell-cell fusion was a three-color assay (7) based on the redistribution of fluorescent probes between effector and target cells upon fusion. The application of three different probes was used to monitor lipid versus cytosolic mixing in the same cell population. Fluorescently labeled gp120-gp41-expressing cells and CD4⁺ cells were cocultured at a 1:10 ratio for 2 h at 37°C in uncoated microwells (MatTek Corp., Ashland, Mass.). Bright-field and fluorescent images were acquired with an Olympus IX70 microscope coupled to a charge-coupled device camera (Princeton Instruments, Trenton, N.J.) with a 40× UplanApo oil immersion objective. Fluorescein isothiocyanate (exciter, BP470-490; beam splitter, DM505; emitter, BA515-550), rhodamine (exciter, BP530-550; beam splitter, DM570; emitter, BA590), and 4′,6-diamidino-2-phenylindole (DAPI) (exciter, D360/40; beam splitter, 400DCLP; emitter, D450/60) optical filter cubes were carefully chosen to avoid spillover when observing the fluorescence of the three dyes. For each sample, three or four different fields were collected, and data were analyzed by overlaying the images using Metamorph software (Universal Imaging Corporation, West Chester, Pa.). The percentage of lipid mixing and cytoplasmic mixing was calculated as 100 times the number of COS-1 cells stained with DiI and calcein divided by the total number of COS-1–HeLa-CD4 conjugates. Although not all COS-1 cells express env since the transfection efficiency is not 100%, env-expressing COS-1 cells are more likely to adhere to HeLa-CD4 cells.Figure ​Figure22 shows a montage of video images taken 2 h following incubation of COS-1 cells expressing wild-type (WT), W(1-5)A, and +DAF env with HeLa-CD4 cells at 37°C. As described in detail in the legend to Fig. ​Fig.2,2, we clearly observed COS-1 cells attached to HeLa-CD4 cells, which showed continuity of all three dyes (CMAC, calcein, and DiI). We know that for +DAF and W(1-5)A env-expressing COS-1 cells, these images do not represent syncytia since even small heterokaryons will show up in the MAGI cell assay (6a), which is based on the transfer of HIV-1 Tat coexpressed with env in COS-1 cells to HeLa-CD4 cells as a result of cell fusion. This transfer induces the expression of a β-galactosidase reporter gene engineered in HeLa-CD4 (MAGI) cells under the control of the viral long terminal repeat promoter (8a). Because the β-galactosidase has been modified to contain a nuclear targeting signal, the nuclei of the resulting heterokaryons stain dark blue with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) in situ. The MAGI assay is extremely sensitive and clearly identifies syncytia as small as two nuclei. Such nuclei were common for the Δ678-682 mutant, which produced syncytia with an average size of ∼5 nuclei (Fig. ​(Fig.3).3). The assay can detect even a few of these fusion events per 100,000 cells. In the MAGI assay, we did not observe any blue nuclei with the W(1-5)A and +DAF constructs, an experiment repeated several times. The three-color assay therefore reveals a distinct phenotype exhibited by the +DAF and W(1-5)A mutant envelope glycoproteins, which form small fusion pores allowing movement of lipids and small molecules (<1,000 Da) but not of large molecules (HIV-1 Tat is about 14 kDa [4b]). Open in a separate windowFIG. 2Three-color assay for WT and mutant HIV-1 gp41s. Simian virus 40-based env expression plasmids (1 μg) containing WT (A to D), W(1-5)A (E to H), and +DAF (I to L) env genes were transfected into COS-1 cells in 35-mm plates using DEAE-dextran (1 μg/ml). At 14 h posttransfection, the cells were replated, and starting at 36 to 48 h posttransfection they were incubated with 20 μM CMAC in Dulbecco modified Eagle medium overnight at 37°C. All constructs expressed similar amounts of envelope glycoprotein on the cell surface (8a). The transfected cells were then washed and incubated in fresh medium for 2 h at 37°C before addition of HeLa-CD4 cells which were labeled in the membrane with DiI and in the cytosol with calcein as previously described (7). The COS-1 cells, labeled with CMAC, were cocultured 1:10 at 37°C for 2 h with HeLa-CD4 cells labeled with DiI and calcein, and images were examined by bright-field microscopy (A, E, and I) and fluorescence microscopy for CMAC staining (B, F, and J), for DiI staining (C, G, and K), and for calcein staining (D, H, and L). CMAC is a fluorescent chloromethyl derivative that freely diffuses through the membranes of live cells. Once inside the cell, this mildly thiol-reactive probe undergoes what is believed to be a glutathione S-transferase-mediated reaction to produce membrane-impermeant fluorescent dye adducts with glutathione, as well as with other intracellular components. Staining of COS-1 cells with CMAC gives rise to bright fluorescence due to reaction with proteins in the perinuclear, endoplasmic reticulum, and Golgi regions, which are immobile, as well as to weaker fluorescence due to the fluorescent glutathione adduct (molecular mass, ∼600 Da) in the cytosol, which is able to diffuse through small fusion pores. The COS-1 cells identified by CMAC staining (B, F, and J) are large and often appear multinuclear, although we do not know whether the round granular structures seen by bright-field microscopy of the COS-1 cells are nuclei or large granules. Panels A to D show one large cell triple stained with CMAC, DiI, and calcein (indicated by a star). DiI is internalized after 2 h at 37°C and appears punctate with nuclear sparing due to its localization in membranes of intracellular organelles. Calcein (465 Da) is evenly distributed throughout the cell (D). One large, granular COS-1 cell (A, left) is only stained with CMAC (B); its lack of staining with DiI (C) and calcein (D) indicates that it has not fused with HeLa-CD4 cells. In panel F, a large structure is seen which seems in continuity with CMAC. However, since the bottom left part of this structure is not in continuity with DiI (G) and calcein (H), it represent two cells. The top right cell (indicated by a star) is in continuity with CMAC, DiI, and calcein. Since COS-1 cells expressing W(1-5)A env do not produce blue nuclei when incubated with MAGI cells (see Fig. ​Fig.3),3), which requires transfer of the 14-kDa HIV-1 Tat protein (see text), we conclude that this COS-1–HeLa-CD4 conjugate represents a phenotype in which small fusion pores form, allowing movement of lipids and small molecules (<1,000 Da) but not of large molecules. The same phenotype is seen with COS-1 cells expressing DAF env: the COS-1–HeLa-CD4 conjugate indicated by a star in panels J, K, and L is in continuity with CMAC, DiI, and calcein but does not allow transfer of HIV-1 Tat (see Fig. ​Fig.33).Open in a separate windowFIG. 3Fusogenic activity of WT and mutant HIV-1 gp41s. The three-color assay was performed as described in the legend to Fig. ​Fig.2.2. Since multiple rounds of fusion may interfere with quantitation in the case of WT and mutant env genes which produce a large number of blue nuclei after 24 h at 37°C (grey bars), incubations were done for 2 h at 37°C. Black bars represent 100 times the number of COS-1 cells stained with DiI and calcein over the total number of COS-1–HeLa-CD4 conjugates measured in the three-color assay. The data are representative of five separate experiments. In each experiment, a total of 30 to 50 COS-1–HeLa-CD4 conjugates were counted. The number of nuclei per syncytium (grey bars) was obtained from the MAGI assay (8a) and represents the ability of HIV-1 Tat to transfer from COS-1 cells to HeLa-CD4 cells.We tallied data from many cell pairs similar to those shown in Fig. ​Fig.22 and plotted the average percentage of COS-1 cells stained with DiI and calcein. Figure ​Figure33 shows the data for the WT and a number of mutants described by Salzwedel et al. (8a). The data fall into three groups, in which the envelope glycoproteins mediate (i) both dye and HIV-1 Tat redistribution (WT, Δ678-682, and SC7), (ii) neither dye nor HIV-1 Tat redistribution (Δ665-682 and +FLAG), or (iii) dye but not HIV-1 Tat redistribution [W(1-5)A and +DAF]. The latter represents a nonexpanding fusion pore phenotype.Dye redistribution induced by WT and mutant gp41s was inhibited by the peptide inhibitors DP178 and C34 (Fig. ​(Fig.4).4). The latter peptide is from the HR2 sequence (residues 628 to 663) which forms the flanking peptide of the heterotrimeric coiled coil in the crystal structure. DP178 is frameshifted 10 amino acids toward the C terminus (residues 638 to 673). The inhibition data indicate that dye redistribution mediated by WT and mutant gp41 molecules is specific for the gp120-gp41-induced fusion reaction and not due to nonspecific transfer. Interestingly, W(1-5)A and SC7 exhibited greater sensitivity than the WT to DP178 inhibition. In the case of C34, inhibition was about the same for the WT and the two mutants. We observed no inhibition by DP178 or C34 of HIV-2 env-mediated fusion at up to 100 nM peptide (data not shown). Open in a separate windowFIG. 4Inhibition of cell-cell fusion by DP178 and C34 peptides. Cell fusion was calculated as a percentage of the control by using the three-color assay method shown in Fig. ​Fig.22 and ​and33 and described in the text for the WT, W(1-5)A, and SC7.Although the crystal structure of the gp41 core (4, 11) is based on the HR1-HR2 coiled coil, it is possible that in intact gp41 the bundle is extended to include amino acids downstream from HR2 and upstream from HR1. Extension of the coiled coil might lead to tilting of the TM anchor, which is presumably important for producing sufficient lipid curvature to form a fusion junction (1). Removal of amino acids 665 to 682 may leave no possibility to form this extended coiled coil. Similarly, insertion of the FLAG sequence, which contains four aspartic acid residues, would presumably insert charged residues into a hydrophobic domain, which could also prevent extension of the coiled coil. The other mutations presumably allow extended coiled-coil formation but reduce its efficiency because of weaker interactions between the amino acids in the extended region. The coiled-coil structure might be so frail in mutant gp41s W(1-5)A and +DAF that it is not present for a sufficient amount of time to create the fusion pore dilation necessary to allow transfer of HIV-1 Tat. Since the Δ678-682 and SC7 proteins are, to a limited extent, capable of inducing syncytium formation and dye transfer, we surmise that they possess intermediate extended coiled-coil-forming propensities.Based on the structural information about the gp41 core (4, 10, 11), it has been proposed that the binding site for the peptide inhibitors is in the HR1 bundle. The C34 and DP178 peptides presumably bind in the same way as the corresponding amino acid sequence regions of the three HR2 helices in the crystal structures. At this position, the peptides would sterically block the regular binding of the HR2 helices to the inner core of HR1 helices and thus prevent formation of the bent-in-half, antiparallel, heterotrimeric coiled-coil structure presumably required to bring the viral and target cell membranes into contact for fusion. Since C34 corresponds to HR2 with no amino acids in the extended region, we do not expect any enhanced inhibitory effect on fusion mediated by the mutant gp41s. Figure ​Figure4b4b shows that this is the case. Since DP178 does contain 10 amino acids downstream from HR2 whose interaction with amino acids upstream from HR1 is weaker in the mutants, we expect greater sensitivity to DP178 inhibition in the mutant proteins. This does seem to be the case, as shown in Fig. ​Fig.44a.The recent high-resolution X-ray crystallographic determination of the structure of the gp41 core from HIV-1 provides well-defined landmarks in the terrain the viral envelope glycoproteins navigate following CD4 and coreceptor-induced conformational changes (5). The structures include neither fusion peptides and TM anchors nor regions between those domains and HR1 and HR2, respectively, which are crucial for fusion activity. Therefore, mutagenesis of those undetermined domains combined with sensitive assays for the activity of the modified proteins will lead to refinement of our thinking about the HIV-1 gp120-gp41 fusion machine.     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. ​(Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man₉GlcNAc₂ precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man₅GlcNAc₂ intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. ​(Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man₃GlcNAc₂ pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. ​(Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc₃Man₉GlcNAc₂; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man₅GlcNAc₂ (Fig. ​(Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man₅GlcNAc₂ substrate (19) (Fig. ​(Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. ​(Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. ​Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc₃Man₉GlcNAc₂ precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man₉GlcNAc₂ precursor into Man₅GlcNAc₂ or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. ​(Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man₅GlcNAc₂ substrate, preventing the formation of hybrid and complex carbohydrates (Fig. ​(Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man₅GlcNAc₂ rather than with the larger Man₉GlcNAc₂ precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man₅GlcNAc₂ we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Cross-Subtype Neutralization Sensitivity despite Monoclonal Antibody Resistance among Early Subtype A,C, and D Envelope Variants of Human Immunodeficiency Virus Type 1

The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.Most effective viral vaccines are thought to provide protection primarily by stimulating neutralizing antibodies (NAbs) to clear cell-free virus (25, 27). Because protection by NAbs requires recognition of common viral epitopes, the extreme genetic diversity of human immunodeficiency virus type 1 (HIV-1) presents a particular challenge to NAb-based vaccine approaches. Therefore, a critical starting point for studies of immune-mediated protection against HIV-1 is a collection of newly transmitted HIV-1 variants, particularly from areas of endemicity, such as sub-Saharan Africa, in order to determine whether vaccines are appropriately targeted to common epitopes from these relevant transmitted strains.During HIV-1 transmission, a bottleneck allows only one or a few variants to be transmitted to a newly infected individual (6, 9, 16, 29, 34, 37, 39), and the sensitivity of these early transmitted strains to antibody-mediated neutralization is therefore of particular interest. Newly transmitted HIV-1 variants have demonstrated significant heterogeneity in their neutralization phenotypes both within and between subtypes (2, 3, 6-8, 11, 13-15, 22, 30, 32, 36). Panels of sexually transmitted HIV-1 envelope variants (based on the envelope gene, env) have been characterized, including subtype B variants from North America, Trinidad, and Europe, subtype C variants from South Africa and Zambia, and subtype A variants from Kenya collected between 1994 and 1996 (2, 14, 15). Here, we characterize an additional 31 envelope variants from 14 subjects with sexually transmitted HIV-1 who were infected in Kenya, where subtypes A, C, and D circulate, between 1993 and 2005 (24, 31).The env genes were cloned from samples drawn 14 to 391 (median, 65) days postinfection from individuals enrolled in a prospective cohort of high-risk women in Mombasa, Kenya (19-21). Demographic characteristics of the subjects are summarized in Table ​Table1;1; the timing of first infection was determined by both HIV-1 serology and HIV RNA testing as described previously (12). All of the subjects were presumably infected by male-to-female transmission and displayed a range of plasma viral loads at the time of env gene cloning (Table ​(Table1).1). For most individuals, full-length env genes were cloned from uncultured peripheral blood mononuclear cell (PBMC) DNA, though for two individuals, clones were obtained from DNA following short-term coculture with donor PBMCs (Table ​(Table1).1). env genes were cloned by single-copy nested PCR with primers and PCR conditions as described previously (4, 17). We tested env genes for their ability to mediate infection by transfecting env plasmid DNA into 293T cells along with an env-deficient HIV-1 subtype A proviral plasmid, Q23Δenv, to make pseudoviral particles (17). More than 80 env clones were obtained from 16 subjects; less than one-half were functional on the basis of the infectivity of pseudoviral particles in a single-round infection of TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health), as observed previously for env genes cloned from proviral sequences (17); a lower fraction of functional env genes have been reported from plasma (18). We focused on the proviral sequences here because they presumably best represent the sequence closest to that of the transmitted strains. The 31 functional env variants are described in Table ​Table11.

TABLE 1.

Demographic characteristics, diversities, gp120 variable-region lengths, numbers of PNGS, and accession numbers of cloned env variants

Comparative Immunogenicity of Subtype A Human Immunodeficiency Virus Type 1 Envelope Exhibiting Differential Exposure of Conserved Neutralization Epitopes

Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.The ability to elicit broadly cross-reactive neutralizing antibodies (NAbs) is likely to be an important component of an effective vaccine to human immunodeficiency virus type 1 (HIV-1). Unfortunately, the HIV-1 envelope (Env)-based vaccines developed to date do not elicit such antibodies. Initial vaccines based on soluble, monomeric gp120 generated antibodies capable of only weakly neutralizing the homologous virus, with a very narrow breadth of cross-reactivity (13, 30, 53). Subsequent modifications to the Env immunogens, including variable loop deletions (15, 20, 31, 34, 35, 61, 64-66), alterations in the glycosylation pattern (4, 10, 11, 14, 30, 43, 55, 56), epitope repositioning (39, 46), the use of consensus Envs (22, 36, 37, 47), and the use of soluble trimeric gp140 molecules as immunogens (1-3, 5, 14, 16, 20, 21, 24, 25) have led to only modest enhancements in NAb breadth or potency. These modified Env immunogens have failed to redirect NAbs from the variable loops to more conserved regions of Env (reviewed in reference 33).Differences in Env structure between HIV-1 subtypes may further hinder efforts to elicit broadly cross-reactive antibodies capable of protecting against transmitted strains worldwide. Most immunogens tested to date have been derived from subtype B Envs. However, there are clear antigenic differences between subtype B strains and the subtype A and C strains that account for most infections worldwide (6, 8, 27, 28, 40, 42). For instance, most transmitted subtype A Envs are resistant to the monoclonal antibodies 2G12, b12, 2F5, and 4E10, either because of alterations in the epitopes for these monoclonal antibodies (MAbs) or because the epitopes are shielded in these Envs (6, 8). It is therefore possible that even NAbs specific for a conserved region of subtype B Envs, such as the CD4 binding site, would not be able to access and neutralize a similar epitope on a subtype A Env.In order to evaluate the immunogenicity of subtype A Envs, which account for ∼25% of global HIV-1 infections (12), we previously investigated the types of antibody responses elicited following gp160 priming and gp140 boosting with immunogens derived from four subtype A Envs in comparison to the subtype B Env SF162 (38). These experiments were also designed to explore whether deriving immunogens from HIV-1 Envs isolated from early in infection would better target NAbs to transmitted strains. Although all of the subtype A-based immunogens and the SF162 immunogen elicited anti-V3 NAbs capable of neutralizing the easy-to-neutralize SF162 pseudovirus, only one of the four immunogens generated homologous NAbs (38). Even immunogens with shorter variable loops or fewer potential N-linked glycosylation sites (PNGS) did not lead to enhanced breadth of neutralization against heterologous subtype A or B Envs (38). However, the four subtype A Envs used in these immunizations were generally neutralization resistant to both plasma samples from HIV-1-infected individuals and to monoclonal antibodies (6), raising the possibility that the poor breadth observed could be related to the shielding of conserved epitopes within these Envs.In order to determine whether using subtype A Env immunogens that do not shield conserved epitopes could improve neutralization breadth, here we performed immunizations with pairs of Env immunogens derived from two individuals acutely infected with subtype A HIV-1. The Envs in each pair were very similar in their amino acid sequences yet differed dramatically in their neutralization phenotype (6, 9) (Fig. ​(Fig.1A).1A). The pair from subject Q461 had a neutralization-resistant Env, {type:entrez-protein,attrs:{text:Q461e2,term_id:123931369,term_text:Q461E2}}Q461e2 (termed {type:entrez-protein,attrs:{text:Q461e2,term_id:123931369,term_text:Q461E2}}Q461e2^R to indicate neutralization resistance), and a neutralization-sensitive Env, {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1 (termed {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1^S to indicate neutralization sensitivity), which was sensitive to neutralization by plasma, 2F5, 4E10, b12, and soluble CD4 (sCD4). We previously demonstrated that the neutralization sensitivity of the {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1^S Env is mediated entirely by two amino acid substitutions in gp41, one in the first heptad repeat and one in the membrane proximal external region (MPER) (9). These mutations led to enhanced exposure of both the CD4 binding site and the MPER (9). From subject Q168, the Env Q168b23^S was sensitive to autologous and heterologous plasma and to the MPER antibodies 2F5 and 4E10 but resistant to b12 and sCD4, while {type:entrez-protein,attrs:{text:Q168a2,term_id:123361966,term_text:Q168A2}}Q168a2^R was weakly neutralized by the MPER antibodies, less sensitive to neutralization by autologous plasma, and resistant to heterologous plasma (6). The {type:entrez-protein,attrs:{text:Q168a2,term_id:123361966,term_text:Q168A2}}Q168a2^R and Q168b23^S Envs contain identical sequences in the MPER region yet have >500-fold differences in neutralization sensitivity to 2F5 and 4E10, indicating that the exposure of the MPER region, rather than the sequence, likely accounts for the enhanced neutralization of the Q168b23^S Env.Open in a separate windowFIG. 1.Analysis of {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1^S gp140 used for immunizations. (A) SDS-PAGE analysis of final preparation of {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1^S gp140 from the GNA capture and DEAE and CHAP columns. Lane 1 contains molecular weight standards, lane 2 the concentrated DEAE flowthrough, and lane 3 the final concentrated protein. The purified {type:entrez-protein,attrs:{text:Q461d1,term_id:123852094,term_text:Q461D1}}Q461d1^S gp140 protein is indicated by an arrow. The sizes of the molecular weight markers (in thousands) are indicated on the left. (B) Binding of purified gp140 subtype A to CD4 as determined by a high-pressure liquid chromatography (HPLC)-based assay. The bottom line represents the protein obtained after the GNA column, and the top line represents purified protein after all three steps. The trimer and monomer peaks are marked. (C) Summary of neutralization characteristics of all four HIV-1 subtype A Env variants used in the immunizations, adapted from reference 6. The pseudovirus is shown in the far left column. IC₅₀ values for plasma sample (left) and monoclonal antibodies (right) are displayed. The autologous plasma samples were taken 3.7 ypi for subject Q461 and 2.6 ypi for subject Q168. The Kenya pool was derived by pooling plasma from 30 HIV-1-infected individuals in Kenya and has been described previously (6).Thus, to directly test whether using Env immunogens that expose conserved epitopes could enhance neutralization breadth immunization, here we immunized with these pairs of related Envs, in which one variant exposes conserved regions, while the other does not. We also compared the specificity of the NAb responses following immunization with these Envs with the specificities of the NAbs that developed during natural infection in the individuals from whom these variants were cloned.     

The Human Immunodeficiency Virus Type 1 Envelope Spike of Primary Viruses Can Suppress Antibody Access to Variable Regions

The human immunodeficiency virus type 1 (HIV-1) envelope spike is a heavily glycosylated trimeric structure in which protein surfaces conserved between different HIV-1 isolates are particularly well hidden from antibody recognition. However, even variable regions on the spike tend to be less antigenic and immunogenic than one might have anticipated for external structures. Here we show that the envelope spike of primary viruses has an ability to restrict antibody recognition of variable regions. We show that access to an artificial epitope, introduced at multiple positions across the spike, is frequently limited, even though the epitope has been inserted at surface-exposed regions on the spike. Based on the data, we posit that restricted antibody access may be the result, at least in part, of a rigidification of the epitope sequence in the context of the spike and/or a highly effective flexible arrangement of the glycan shield on primary viruses. Evolution of the HIV envelope structure to incorporate extra polypeptide sequences into nominally accessible regions with limited antibody recognition may contribute to reducing the magnitude of antibody responses during infection and allow the virus to replicate unhindered by antibody pressure for longer periods.     

Antibody Neutralization-Resistant Primary Isolates of Human Immunodeficiency Virus Type 1

Although typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1⁺ human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869–6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.     

Broadly Neutralizing Monoclonal Antibodies 2F5 and 4E10 Directed against the Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Protect against Mucosal Challenge by Simian-Human Immunodeficiency Virus SHIVBa-L

The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIV_Ba-L. In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.Eliciting broadly neutralizing antibodies is an important goal of HIV vaccine design efforts, and the study of broadly neutralizing monoclonal antibodies (bnMAbs) can assist in that goal. Human bnMAbs against both gp120 and gp41 of the HIV-1 envelope spike have been described. Three bnMAbs to gp41, 2F5, 4E10, and Z13e1, have been identified and shown to recognize neighboring linear epitopes on the membrane proximal external (MPER) region of gp41 (3, 24, 25, 37, 47). In a comprehensive cross-clade neutralization study by Binley et al., 2F5 neutralized 67% and 4E10 neutralized 100% of a diverse panel of 90 primary isolates (2). Similar broad neutralization was seen against sexually transmitted isolates cloned from acutely infected patients (22). More recently, a comprehensive study showed that 2F5 neutralized 97 isolates from a 162-virus panel (60%) and that 4E10 neutralized 159 isolates (98%) (41). Although less potent, the monoclonal antibody Z13, isolated from an antibody phage display library derived from a bone marrow donor whose serum was broadly neutralizing (47), has cross-clade neutralizing activity. Z13e1 is an affinity-enhanced variant of the earlier-characterized MAb Z13 that is directed against an access-restricted epitope between and overlapping the epitopes of 2F5 and 4E10. Both MAbs 2F5 and 4E10 were originally obtained as IgG3 antibodies in hybridomas derived from peripheral blood mononuclear blood lymphocytes (PBMCs) of HIV-1-seropositive nonsymptomatic patients and were later class switched to IgG1 to enable large-scale manufacturing and to prolong in vivo half-life (3, 6, 32).Despite the interest in the MPER as a vaccine target, there is limited information on the ability of MPER antibodies to act antivirally in vivo either in established infection or prophylactically. A study using the huPBL-SCID mouse model showed limited impact from 2F5 when the antibody was administered in established infection (31). Passive administration of 2G12, 2F5, and 4E10 to a cohort of acutely and chronically infected HIV-1 patients provided little direct evidence of 2F5 or 4E10 antiviral activity, whereas the emergence of escape variants indicated unequivocally the ability of 2G12 to act antivirally (18, 39). Indirect evidence did, however, suggest that the MPER MAbs may have affected virus replication, as indicated by viral rebound suppression in a patient known to have a 2G12-resistant virus prior to passive immunization (39). Another study of 10 individuals passively administered 2G12, 2F5, and 4E10 before and after cessation of combination antiretroviral therapy (ART) showed similarly that 2G12 treatment could delay viral rebound, but antiviral activity by 2F5 and 4E10 was not clearly demonstrated (21). In prophylaxis, an early 2F5 passive transfer study with chimpanzees suggested that the antibody could delay or lower the magnitude of primary viremia following HIV-1 challenge (7). A study using gene transfer of 2F5 in a humanized SCID mouse model suggested that continuous plasma levels of approximately 1 μg/ml of 2F5 may significantly reduce viral loads in LAI- and MN-challenged mice (34). Protection studies of rhesus macaques using simian-human immunodeficiency virus SHIV_89.6PD challenge did not provide definitive direct evidence for MPER antibody-mediated protection. One of three animals was protected against intravenous (i.v.) challenge when 2F5 was administered in a cocktail with HIVIG and 2G12 (19), but all three animals treated with 2F5 alone at high concentration became infected. In a vaginal challenge study with SHIV_89.6PD (20), four of five animals were protected with a cocktail of HIVIG, 2F5, and 2G12, but a 2F5/2G12 combination protected only two of five animals. Further protection studies have used MPER MAbs in combination with other MAbs, leaving the individual contributions of these antibodies uncertain (1, 8).In our previous studies, we successfully used the SHIV/macaque model to demonstrate neutralizing antibody protection against mucosal challenge, and we have begun to explore how that protection is achieved (12, 30). Here, we conducted a protection study with the two broadly neutralizing MPER-directed antibodies 2F5 and 4E10. We show that the antibodies can prevent viral infection and thereby support the MPER as a vaccine target.     

Neutralizing Antibody Responses in Macaques Induced by Human Immunodeficiency Virus Type 1 Monovalent or Trivalent Envelope Glycoproteins

A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb). In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs) would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q), was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B) and 14/00/4 (subtype F), both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q). The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.     

A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.     

A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Common Themes of Antibody Maturation to Simian Immunodeficiency Virus, Simian-Human Immunodeficiency Virus, and Human Immunodeficiency Virus Type 1 Infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.     

Introduction of Exogenous Epitopes in the Variable Regions of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Effect on Viral Infectivity and the Neutralization Phenotype

In this study we examined whether human immunodeficiency virus type 1 (HIV-1) is equally susceptible to neutralization by a given antibody when the epitope of this antibody is introduced at different positions within the viral envelope glycoprotein (Env). To this end, we introduced two exogenous “epitope tags” at different locations within three major Env regions in two distinct HIV-1 isolates. We examined how the introduction of the exogenous epitopes affects Env expression, Env incorporation into virions, Env fusogenic potential, and viral susceptibility to neutralization. Our data indicate that even within the same Env region, the exact positioning of the epitope impacts the susceptibility of the virus to neutralization by the antibody that binds to that epitope. Our data also indicate that even if the same epitope is introduced in the exact same position on two different Envs, its exposure and, as a result, the neutralization susceptibility of the virus, can be very different. In contrast to the findings of previous studies conducted with HIV-1 isolates other than those used here, but in agreement with results obtained with simian immunodeficiency virus, we observed that tagging of the fourth variable region of Env (V4) did not result in neutralization by the anti-tag antibodies. Our data indicate that epitopes in V4 are not properly exposed within the functional HIV-1 trimeric Env spike, suggesting that V4 may not be a good target for vaccine-elicited neutralizing antibodies.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is expressed as a heavily glycosylated peptide of approximately 160 kDa (gp160), which is cleaved intracellularly into two noncovalently associated subunits: an extracellular subunit (gp120), responsible for CD4 and coreceptor (primarily CCR5 and/or CXCR4) binding, and a transmembrane subunit (gp41) that mediates fusion between viral and host cell membranes. Based on amino acid sequence homology analysis of gp120s derived from diverse HIV-1 isolates, gp120 is divided into five “constant” regions (C1 to C5) and five “variable” regions (also called “loops,” because most of them have cysteines in the N and C termini that form disulfide bonds). Despite their extensive amino acid variability, the variable loops of gp120 play central roles during the entry of the virus into the cell, for instance, by directly or indirectly modulating the interaction of Env with coreceptor molecules on the target surfaces during virus-cell fusion. They also offer protection from neutralizing antibodies (NAbs) by various mechanisms. The variable loops themselves are targets of NAbs, and during infection, the replicating virus accumulates mutations in the variable regions that allow it to escape the action of anti-variable loop-directed NAbs, while at the same time the variable loops are positioned within the Env trimer so that they prevent, or minimize, the binding of NAbs to more-conserved epitopes, such as the receptor and coreceptor binding sites (4, 5, 12, 15, 20, 23, 25, 27, 31).HIV-1 strains display distinct neutralization phenotypes. Some isolates, such as SF162, are generally susceptible to NAbs that bind to many distinct regions of Env, including the variable regions, while other isolates, such as YU2 or JRFL, are generally resistant to neutralization by the same NAbs (1). It has been proposed that irrespective of the overall neutralizing phenotype of HIV-1 isolates, the binding of only a single antibody per Env trimer on the virion surface can lead to neutralization, when all Env trimers present on the virion surface are bound by at least one antibody (32). This important observation also implies that the epitope specificity of an antibody may not be as important for neutralization as its ability to bind to its target within the trimeric Env structure. In fact, antibodies to diverse regions of Env, such as V1, V2, V3, and the receptor and coreceptor binding sites, can all neutralize HIV-1 (1, 3, 6, 8, 10, 18, 20, 23, 25, 27, 29, 30).In many cases, a given isolate will not be equally susceptible to neutralization by NAbs that bind to different Env regions, for example, the V3 loop and the CD4-binding site (CD4-BS). Whether differences in the neutralizing potentials of two antibodies that bind to distinct epitopes on HIV-1 Env are due to differences in the binding affinities of the two antibodies or whether they occur because the viruses are intrinsically more susceptible to NAbs that bind certain epitopes and not others (i.e., the relative importance of the various regions of Env in Env function and virus neutralization sensitivity differs) is not yet fully understood. One way to address these issues is to introduce small non-HIV Env amino acid sequences (tags) that are targets of known monoclonal antibodies (MAbs) at various positions within the viral Env and to examine how the placement of the same epitope at different positions within Env affects the neutralization phenotype of the virus.Foreign epitopes have been introduced into the variable regions of HIV and simian immunodeficiency virus (SIV) Envs, and their effects on viral neutralization potential have been examined (14, 19, 22, 33). Yang and colleagues (33) introduced the FLAG epitope into the V4 regions of three HIV-1 isolates (YU2, JRFL, and HxB2) displaying distinct neutralization phenotypes in response to anti-HIV NAbs; they found that all three pseudotyped viruses were equivalently neutralized by an anti-FLAG MAb. One important implication of that study is that neutralization-resistant isolates, such as YU2 or JRFL, are not intrinsically more resistant to neutralization than more-susceptible isolates, such as HxB2, so long as the antibody binds to its epitope on the functional virion-associated Env spike. A second implication is that since the FLAG epitope was exposed in the V4 loops of all three isolates, the V4 loop could theoretically be a good target for vaccine-elicited antibodies. In contrast, Pantophlet et al. (19) introduced the HA tag into various regions of the JRCSF (neutralization-resistant) and HxB2 (neutralization-sensitive) isolates and reported that JRCSF was intrinsically more resistant than HxB2 to anti-HA antibodies. This observation implies, therefore, that some HIV-1 strains (primary, neutralization-resistant strains) have developed mechanisms that limit the accessibility of multiple Env regions, including variable regions, to antibodies developed during infection. Laird and Desrosiers (14) introduced the FLAG epitope into two positions within each of the V1, V2, and V4 loops of SIV239 and SIV316. They reported that the functionality of Env was differentially affected by the precise location of the exogenous tag sequence within the variable loops examined. Importantly, and in contrast to what was reported for the HIV-1 isolates mentioned above, the SIV239 variants containing a V4 FLAG epitope were not neutralized by an anti-FLAG MAb. It appeared, however, that the FLAG epitope was not well exposed on the trimeric Env when introduced into the V4 loop of SIV but was exposed when introduced into the V1 loop of the same virus. Potentially, this means that the V4 loop is differentially exposed in the context of the HIV-1 and SIV Envs.The FLAG epitope (DYKDDDDK) is highly charged. Therefore, it is possible that the effect on Env function and epitope exposure could differ if a different exogenous epitope were inserted instead of FLAG. Here we examined the effect of variable loop tagging on the Env functions and viral neutralization phenotypes of two primary HIV-1 clade B isolates, SF162 (CCR5 tropic) and SF33 (CXCR4 tropic), using two exogenous epitopes (FLAG and hemagglutinin [HA] tags) positioned at multiple locations within the V1, V2, and V4 loops. By placing the same tag in several regions within each loop, we investigated the accessibilities of various parts of the same loop to a given NAb. By using two tags that differ significantly in amino acid composition (FLAG tag, DYKDDDDK; HA tag, YPYDVPDYA), we aimed at distinguishing between the effects of amino acid composition and the positioning of the tag on Env function and overall epitope exposure. Finally, identical evaluations of R5 and X4 Envs may provide information about the relative roles played in neutralization by variable loops in Envs displaying distinct coreceptor usage. We report that both the amino acid sequence and the position of the tag within and among the variable loops greatly affected the functionality of Env. In contrast to previous observations made with other HIV-1 Envs (33) but in agreement with what was reported for the SIV239 Env (14), we observed that tagging of the V4 loops of SF162 and SF33 did not render these isolates susceptible to neutralization by the corresponding anti-tag MAbs.