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Human immunodeficiency virus type-1 (HIV-1) antigenic variation poses a great challenge for vaccine immunogen design to elicit broadly neutralizing antibodies (bNAbs). Over the last 10-15 years, great progress has been made to understand the conserved sites of sensitivity on HIV envelope glycoprotein spikes targeted by bNAbs. Plasma neutralization mapping and monoclonal antibody isolation efforts have revealed five major conserved epitope clusters. Most of this work has focused on subtype B and C-infected Caucasian or African donors. It is not clear if the same epitopes and epitope rank order preferences are also true in donors infected with different HIV-1 subtypes and with different racial backgrounds. To investigate this point, in this study we report the first attempt to profile the bNAb specificities of CRF01_AE-infected Malaysian plasmas. We first measured neutralization titers of 21 plasmas against a subtype A, B, and AE pseudovirus panel. This revealed that 14% (3 of 21) plasmas had cross-clade breadth. Focusing on the cross-neutralizing plasma P9, we used AE and JR-FL mutant pseudoviruses, gp120 monomer interference, and native polyacrylamide gel electrophoresis to better understand the neutralization specificity. P9 demonstrates CD4-binding-site specificity with trimer dependence and D368 independence.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts., Author summary Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV’s surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their ’demands’ for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts.

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation, and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies, but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C-433C (DS) variant, specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like particle and soluble formats providing a new generation of vaccine antigens.

Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1., Author Summary HIV-1 remains a significant global health threat, with no effective vaccine against the virus currently available. Since traditional vaccine design efforts have had limited success, much effort in recent years has focused on gaining a better understanding of the ways select individuals are able to effectively neutralize the virus upon natural infection, and to utilize that knowledge for the design of optimized vaccine candidates. Primary emphasis has been placed on characterizing the antibody arm of the immune system, and specifically on antibodies capable of neutralizing the majority of circulating HIV-1 strains. Various experimental techniques can be applied to map the epitope targets of these antibodies, but more recently, the development of computational methods has provided an efficient and accurate alternative for understanding the complex antibody responses to HIV-1 in a given individual. Here, we present the next generation of this computational technology, and show that these new methods have significantly improved accuracy and confidence, and that they enable the interrogation of biologically important questions that can lead to new insights for the design of an effective vaccine against HIV-1.

The HIV-1 spike is composed of three protomeric units, each containing a peripheral gp120 and a transmembrane gp41 subunit. Binding to the CD4 and the chemokine receptors triggers them to mediate virus entry into cells by membrane fusion. The spikes also represent the major target for neutralizing antibodies (Abs) against the virus. We have studied how two related broadly neutralizing Abs, PG9 and PG16, react with the spike. Unexpectedly, this also suggested how the functions of the individual protomers in the spike depend on each other. The Abs have been shown to bind the V1/V2 loops of gp120, located at the top of the spike. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we show that only single Abs or antigen-binding fragments could bind to the spikes of HIV-1 virus-like particles. Apparently, binding to one gp120 sterically interferes with binding to the other two subunits in the spike top. Despite this constraint, all of the protomers of the spike became resistant to CD4 binding and subsequent formation of the coreceptor binding site. These activities were measured by monitoring the sequential complex formation of the spike first with Abs and then with soluble 2d- or 4d-CD4 or with soluble CD4 and the CD4 inducible coreceptor binding site Ab 17b in BN-PAGE. The inhibition of the spike by single-Ab binding suggested that the activation reactions of the individual protomeric units are linked to each other in a coordinated activation process.

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen-host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro . The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.

HIV-1 viruses and virus-like particles (VLPs) bear nonnative “junk” forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. “Trimer VLPs” may be useful neutralizing antibody immunogens.

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.

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.

Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.

Insights into the process of HIV-1 neutralization may assist rational vaccine design. Here, we compared antibody neutralization against the JR-FL primary isolate and trimer binding affinities judged by native PAGE. Monovalent Fab-trimer binding and neutralization showed a direct quantitative relationship, implying that neutralization begins as each trimer is occupied by one antibody. At saturation, three Fab or soluble CD4 molecules engaged each trimer. In contrast, a maximum of one soluble CD4 molecule bound to functional SIV trimers with a truncated a gp41 tail. Remarkably, soluble CD4 was found to trigger dramatic enhancement of this virus. Unlike Fabs, a quantitative correlation between JR-FL trimer binding and neutralization was unclear for some, but not all IgGs, as neutralization was markedly increased, but trimer affinity was largely unchanged. In addition, only one molecule of certain gp41-specific IgGs appeared to be able to bind each trimer. We discuss the implications of these findings in weighing the relative contributions of size, multivalent binding and other possible effects of IgGs to explain their increased potency.

To assess the potential of native Envelope glycoprotein (Env) trimers as neutralizing antibody vaccines, we immunized guinea pigs with three types of VLPs and soluble gp120. Particles included “SOS-VLPs” (bearing disulfide-shackled functional trimers), “UNC-VLPs” (bearing uncleaved nonfunctional Env) and “naked VLPs” (bearing no Env). The SOS-VLPs were found to have a density of about 27 native trimers per particle, approximately twice that of live inactivated HIV-1 preparations. As immunogens, UNC- and SOS-VLP rapidly elicited anti-gp120 antibodies focused on the V3 loop and the gp120 coreceptor binding site. Reactivity to the gp41 immunodominant domain was absent in SOS-VLP sera, presumably because gp120–gp41 association is stabilized, effectively covering this epitope. Gp120-immune sera reacted with the receptor binding sites of gp120 and were less focused on the V3 loop. Some Env-VLP sera neutralized primary isolates at modest titers. The measurement of neutralization was found to be affected by the cell lines used. Depending on the assay particulars, non-Env specific antibodies in VLP sera could enhance infection, or nonspecifically neutralize. However, a neutralization assay using TZM-BL cells was essentially clear of these effects. We also describe a native trimer binding assay to confirm neutralization activity in a manner that completely eliminates nonspecific effects. Overall, our data suggests that Env-VLP sera were primarily focused on nonfunctional forms of Env on VLP surfaces, possibly gp120/gp41 monomers and not the trimers. Therefore, to make progress toward a more effective VLP-based vaccine, we will need to find ways to refocus the attention of B cells on native trimers.

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine., Author Summary Here we show that native HIV-1 Env spikes expressed in a natural membrane context can induce potent tier 2 nAbs in rabbits. These antibodies reacted exclusively with epitopes present on these trimers and not with isolated Env subunits. Intriguingly, the neutralizing sera were found to take advantage of natural gaps in the carbohydrate defenses of Env spikes of the vaccine strain. Some sera were able to neutralize heterologous isolates, provided that a key, regulating glycan was removed. Overall, these findings suggest that native, membrane-expressed trimers hold promise for further development as vaccine candidates. In the future, by adapting our current findings, we might be able to encourage nAb development to key conserved sites by introducing additional, targeted gaps in the trimer's glycan shell. We suggest that the rare ability to predictably induce potent autologous neutralizing antibodies to field isolates, as we report here, provides a foundation for exploring new strategies aimed at inducing neutralization breadth which is widely expected to be essential for vaccine-induced protection.

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIVSF162P4, and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, ΔV2gp140 (lacking the crown of the V2 loop), ΔV3gp140 (lacking the crown of the V3 loop), and ΔV2ΔV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and ΔV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and ΔV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by ΔV3gp140 or ΔV2ΔV3gp140. In contrast, the SHIVSF162P4-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.

Understanding the nature of neutralization may provide information for crafting improvements in HIV vaccines. Using JR-FL as a prototype primary pseudovirus, we first investigated anti-HIV monoclonal antibodies (mAbs) in several neutralization formats designed to elucidate the timing of neutralization. MAb b12 was most effective before receptor binding, 2G12 neutralized effectively even after CD4 binding, and X5 and a V3 loop mAb (LE311) were inactive in a standard format but were induced by sCD4. Consistent with this latter finding, native PAGE indicated that X5 and V3 mAb binding to Envelope trimers was dependent on sCD4 binding. In contrast, 2F5 and 4E10 were active even post-CD4/CCR5 engagement. We next analyzed the neutralization mechanism of a panel of HIV+ donor plasmas of various potencies. All mediated high levels of post-CD4 neutralization that was not associated with activity in the standard format. None, however, neutralized effectively in the post-CD4/CCR5 format, suggesting that 2F5/4E10-like Abs were absent or at low concentrations. Finally, we analyzed a non-neutralizing plasma spiked with mAbs b12, 2G12 or 2F5, which resulted in increases in neutralization titers consistent with the activities of the mAbs. We conclude that these methods, together with other mapping approaches, may provide a better understanding of neutralization that could be useful in vaccine research.

SummaryBroadly neutralizing monoclonal antibodies to HIV-1 are rare but invaluable for vaccine design. 4E10 is the broadest neutralizing antibody known and recognizes a contiguous and highly conserved epitope in the membrane-proximal region of gp41. The crystal structure of Fab 4E10 was determined at 2.2 Å resolution in complex with a 13-residue peptide containing the gp41 core epitope (NWFDIT). The bound peptide adopts a helical conformation in which the key contact residues, TrpP672, PheP673, IleP675, and ThrP676, map to one face of the helix. The peptide binds in a hydrophobic pocket that may emulate its potential interaction with the host cell membrane. The long CDR H3 of the antibody extends beyond the bound peptide in an orientation that suggests that its apex could contact the viral membrane when 4E10 is bound to its membrane-proximal epitope. These structural insights should assist in the design of immunogens to elicit 4E10-like neutralizing responses.

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade.

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol . 74: 627-643, 2000). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant’s infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor, typically CCR5. Here we provide evidence that purified gp120 JR-FL –CD4–CCR5 complexes exhibit an epitope recognized by a Fab (X5) obtained by selection of a phage display library from a seropositive donor with a relatively high broadly neutralizing serum antibody titer against an immobilized form of the trimolecular complex. X5 bound with high (nM) affinity to a variety of Envs, including primary isolates from different clades and Envs with deleted variable loops (V1, -2, -3). Its binding was significantly increased by CD4 and slightly enhanced by CCR5. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D, E, F, and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore, X5 inhibited cell fusion mediated by Envs from R5, X4, and R5X4 viruses. Of the five broadly cross-reactive HIV-1-neutralizing human monoclonal antibodies known to date, X5 is the only one that exhibits increased binding to gp120 complexed with receptors. These findings suggest that X5 could possibly be used as entry inhibitor alone or in combination with other antiretroviral drugs for the treatment of HIV-1-infected individuals, provide evidence for the existence of conserved receptor-inducible gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-1-infected patients, and have important conceptual and practical implications for the development of vaccines and inhibitors.

The native, fusion-competent form of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex is a trimeric structure composed of three gp120 subunits and three gp41 subunits; the receptor-binding (CD4 and coreceptor) sites are located in the gp120 moieties, and the fusion peptides are located in the gp41 components (10, 33, 34, 52, 69, 78, 83). In the generally accepted model of HIV-1 fusion, the sequential binding of gp120 to CD4 and a coreceptor induces a series of conformational changes in the gp41 subunits, leading to the insertion of the fusion peptides into the host cell membrane in a highly dynamic process (14, 31, 39, 59, 68, 72, 81, 84, 91). The associations between the six components of the fusion-competent complex are maintained via noncovalent interactions between gp120 and gp41 and between the gp41 subunits (52, 84). These interactions are relatively weak, making the fusion-competent complex unstable. This instability perhaps facilitates the conformational changes in the various components that are necessary for the fusion reaction to proceed efficiently, but it greatly complicates the task of isolating the native complex in purified form. Put simply, the native complex falls apart before it can be purified, leaving only the dissociated subunits. One reason it would be desirable to produce the native HIV-1 envelope complex is to explore its potential as an immunogen, perhaps after modification to improve its exposure of critical neutralization epitopes. The limited neutralizing-antibody response to HIV-1 in infected people is directed at the native complex and is probably raised against it (6, 40, 49, 51); P. W. H. I. Parren, D. R. Burton, and Q. J. Sattentau, Letter, Nat. Med. 3:366, 1997). In contrast, the isolated subunits have not proven efficient at inducing relevant neutralizing antibodies (reviewed in references 6, 49, and 51). We and others are therefore attempting to make more-stable forms of the envelope glycoprotein complex that better mimic the native structure. Usually, these efforts have focused on making various forms of soluble gp140 glycoproteins which contain gp120 but only the ectodomain of gp41 (4, 11, 13, 17, 19-21, 57, 66, 76, 85-87, 90). An approach to resolving the instability of the native complex is to remove the cleavage site that naturally exists between the gp120 and gp41 subunits. Doing so means that proteolysis of this site does not occur, leading to the expression of gp140 glycoproteins in which the gp120 subunit is covalently linked to the gp41 ectodomain (gp41ECTO) by means of a peptide bond (2, 3, 16-18). Such proteins can be oligomeric, sometimes trimeric (11, 16-21, 54, 66, 85-87, 90). However, it is not clear that they truly represent the structure of the native, fusion-competent complex in which the gp120-gp41 cleavage site is fully utilized. Hence the receptor-binding properties of uncleaved gp140 (gp140UNC) proteins tend to be impaired, and nonneutralizing antibody epitopes are exposed on them that probably are not accessible on the native structure (4, 6, 28, 60, 90). We have taken an alternative approach to the problem of gp120-gp41 instability, which is to retain the cleavage site but to introduce a disulfide bond between the gp120 and gp41ECTO subunits (4, 57). Properly positioned, this intermolecular disulfide bond forms efficiently during envelope glycoprotein (Env) synthesis, allowing the secretion of gp140 proteins that are proteolytically processed but in which the association between the gp120 and gp41ECTO subunits is maintained by the disulfide bond. Here we show that the gp41-gp41 interactions are unstable in the SOS gp140 protein, which is expressed and purified primarily as a monomer. In contrast, gp140UNC proteins—with or without the SOS cysteine substitutions—are multimeric, implying that cleavage of the peptide bond between gp120 and gp41 destabilizes the native complex. Despite being monomeric, the purified and unpurified forms of SOS gp140 are better antigenic structural mimics of the native, fusion-competent Env structure than are the corresponding gp120 or gp140UNC proteins. This may be because the presence and orientation of gp41ECTO occludes certain nonneutralization epitopes on SOS gp140 while preserving the presentation of important neutralization sites. This explanation is consistent with immunoelectron microscopy studies of the protein. Unexpectedly, proteolytically mature, but variable-loop-deleted, SOS gp140 glycoproteins have enhanced oligomeric stability, so these molecules warrant further study for their structural and immunogenic properties.

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Molecular and structural characterization is reported for a new broad and potent monoclonal antibody against HIV that binds to an epitope bridging the gp41 and gp120 subunits — the antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. This paper describes a broadly neutralizing HIV-specific monoclonal antibody that binds with high potency to a novel HIV-1 envelope glycoprotein epitope. Molecular and structural characterization of the new antibody, named 35O22, show that it is specific for a new site of vulnerability made up of amino acids and glycans bridging the gp41 and gp120 subunits. The antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. Serologic analysis indicates that antibodies to this newly recognized site of vulnerability are commonly elicited by natural infection, raising the prospect that in may be a promising potential vaccine target. The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50)

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

The effect on humoral immune responses of highly active antiretroviral therapy (HAART) commenced during primary or chronic human immunodeficiency virus type 1 (HIV-1) infection was investigated. HAART inhibited the development of anti-gp120 antibodies when initiated during primary infection and could sometimes reduce antibody titers in patients treated within 2 years of HIV-1 infection. Conversely, antibody responses in patients infected for several years were less sensitive to HAART. Administering HAART during primary infection usually did not substantially affect the development of weak neutralizing antibody responses against autologous virus. However, 2 patients treated very early after infection did not develop neutralizing responses. In contrast, 3 of 4 patients intermittently adherent to therapy developed autologous neutralizing antibodies of unusually high titer, largely coincident with brief viremic periods. The induction of strong neutralizing antibody responses during primary HIV-1 infection might require the suppression of virus replication by HAART, to allow for the recovery of immune competency, followed by exposure to native envelope glycoproteins.

The relationship between human immunodeficiency virus (HIV) type 1 replication and CD4+ T cell function was examined. T lymphocyte proliferation in response to both HIV-1 antigens and recall antigens was measured in HIV-1-infected individuals before and after they received highly active antiretroviral therapy (HAART). No correlation was observed between baseline viral load or CD4+ T cell count and the T cell proliferative response to HIV-1 Gag. Suppression of viremia was not associated with an increase in T cell proliferative responses. Emergence of viral replication during short periods of intermittent therapy promoted generalized activation of T helper lymphocytes, manifested by increased T cell proliferative responses to HIV-1 Gag and recall antigens. Recovery of CD4+ T cell responses occurred in some individuals who initiated HAART years after infection and who were intermittently adherent to drug treatment. Thus, CD4+ T cell responses can sometimes be regenerated if viral load is suppressed to allow some immune recovery and if antigenic stimulation is later provided.

Antibody responses are often considered to play only a limited role in controlling viremia during chronic infections with human or simian immunodeficiency virus (SIV). We investigated this by determining the effect of passively infused antibody on plasma viremia in infected rhesus macaques. The emphasis of the study was to understand the mechanism(s) underlying any observed effects. We infused serum immunoglobulins (SIVIG) purified from SIVmac251-infected macaques into other SIVmac251-infected macaques. The rapid progressor recipients had high viral loads but negligible titers of antibodies to SIV. Thus, we could significantly increase antibody titers with exogenous SIVIG. Despite restoring anti-SIV titers to levels typical of macaques with a normal disease course, SIVIG had only a modest effect on plasma SIV RNA and cell-associated viral load; the maximum, transient, reduction was threefold. The decrease in plasma RNA commenced within 1–2 h of SIVIG infusion, the nadir was at 12 h, and then a rebound occurred. A two- to threefold drop in cell-associated viral RNA was simultaneous with the decrease in plasma RNA. The kinetics of the viremia changes are inconsistent with neutralization of new cycles of infection. More likely, perhaps unexpectedly, is that infused antibodies killed SIV-infected cells, via an effector mechanism such as antibody-dependent cellular cytotoxicity.

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627–643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.

Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels for 3 or more years. However, adherence to complex drug regimens can prove problematic, and subjects may temporarily discontinue HAART for variable periods. We studied 6 HIV-1-infected individuals who stopped therapy. Off HAART, levels of viremia were suppressed to fewer than 500 copies/mL in 2 subjects for more than 12 and more than 24 months, respectively, and in 1 subject for 4 months on 1 occasion. Three subjects failed to contain plasma viremia. Broad and strong HIV-1-specific immune responses were detected in subjects with prolonged suppression of viral replication. This longitudinal study suggests that containment of HIV-1 replication to low or undetectable levels after discontinuation of HAART is associated with strong virus-specific immune responses. Boosting of HIV-1-specific immune responses should be considered as an adjunctive treatment strategy for HIV-1-infected individuals on HAART.

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.

We studied how combination antiviral therapy affects B cell abnormalities associated with HIV-1 infection, namely elevated circulating immunoglobulin (Ig)G antibody-secreting cell (ASC) frequencies and hypergammaglobulinemia. Within a few weeks of starting antiviral therapy, there is a marked decline in IgG-ASC frequency in both acutely and chronically infected people, whereas the hypergammaglobulinemia often present during chronic infection is more gradually resolved. These reductions are sustained while HIV-1 replication is suppressed. HIV-1 antigen–specific B cell responses are also affected by therapy, manifested by a rapid decline in circulating gp120-specific ASCs. Anti-gp120 titers slowly decrease in chronically infected individuals and usually fail to mature in acutely infected individuals who were promptly treated with antiretroviral therapy. Long-term nonprogressors have high titer antibody responses to HIV-1 antigens, but no detectable gp120-specific IgG-ASC, and normal (or subnormal) levels of total circulating IgG-ASC. Overall, we conclude that HIV-1 infection drives B cell hyperactivity, and that this polyclonal activation is rapidly responsive to decreases in viral replication caused by combination antiviral therapy.

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4 + cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses.

The binding of a panel of monoclonal antibodies to V1, V2, and V3 loop-deleted HIV-1 gp120 was studied by competition analysis. Most of the previously defined relationships between gp120 epitopes were preserved on the variable loop-deleted protein, although interactions between some epitopes were dependent on the presence of the V1, V2, and V3 loops. Enzymatic deglycosylation of the variable loop-deleted protein only minimally altered the binding of most antibodies examined. Thus, a carbohydrate-deficient, conserved HIV-1 gp120 core can be produced that has a structure closely approximating that of the full-length, correctly folded gp120 monomer.

The avidity of antibodies for antigens can be measured by determining what remains bound after exposing the antibody-antigen complex to a chaotropic agent such as urea. This method has been gaining popularity for assessing the immune response to the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 (or its counterpart from simian immunodeficiency virus), during natural infection or after subunit vaccination. High-avidity antibodies have been considered to be a possible correlate of protection. We have examined the avidity assay to determine what it, in fact, measures. First, we studied the development of the anti-gp120 response in seroconverting individuals. Urea elution reduced the polyclonal anti-gp120 titers by 3- to 10-fold. After allowing for the consequent reduction in assay sensitivity, there was no obvious change in the rate of development of the high-avidity and unfractionated antibody responses. Furthermore, in the one individual who developed a strong autologous, virus-neutralizing response, the appearance of neutralizing antibodies and high-avidity antibodies did not coincide. Antibodies to the V3 loop, when present, comprised a major fraction of the polyclonal response that survives urea elution. We next examined the effect of urea elution on the binding to gp120 of a panel of monoclonal antibodies (MAbs). Urea treatment preferentially eluted MAbs to discontinuous rather than continuous epitopes, independent of their affinities. Furthermore, these patterns of epitope stability were unaltered by the presence of polyclonal anti-gp120 antibodies. As most broadly neutralizing anti-gp120 antibodies recognize discontinuous epitopes, this skewing effect must be taken into account when interpreting studies using polyclonal sera.

To test whether antibodies that are neutralizing or nonneutralizing for human immunodeficiency virus type 1 (HIV-1) primary isolates can be distinguished by their affinities for the oligomeric envelope glycoproteins, we selected HIV-1(JR-FL) as a model primary virus and a panel of 13 human monoclonal antibodies (MAbs) and evaluated three parameters: (i) half-maximal binding to recombinant monomeric envelope, gp120(JR-FL); (ii) half-maximal binding to oligomeric envelope of HIV-1(JR-FL) expressed on the surface of transfected 293 cells; and (iii) neutralization of HIV-1(JR-FL) in a peripheral blood mononuclear cell-based neutralization assay. Two conclusions can be drawn from these experiments. First, we confirm that antibody interactions with monomeric gp120 do not predict primary virus neutralization. Second, we show that neutralization correlates qualitatively with the relative affinity of an antibody for the oligomeric envelope glycoproteins, at least for HIV-1(JR-FL).

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.

In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less “triggered” conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.