HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound "closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature "opening" of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site.

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis.

HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens.IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

Cryo-EM Structure of Full-length HIV-1 Env Bound With the Fab of Antibody PG16.

The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a "closed" conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain-the "SOSIP" construct-has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env "spike" and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

Molecular Mechanism of HIV-1 Entry.

HIV-1 envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] attaches the virion to a susceptible cell and induces fusion of viral and cell membranes to initiate infection. It interacts with the primary receptor CD4 and coreceptor (e.g., chemokine receptor CCR5 or CXCR4) to allow viral entry by triggering large structural rearrangements and unleashing the fusogenic potential of gp41 to induce membrane fusion. Recent advances in structural biology of HIV-1 Env and its complexes with the cellular receptors have revealed molecular details of HIV-1 entry and yielded new mechanistic insights. In this review, I summarize our latest understanding of the HIV-1 membrane fusion process and discuss possible pathways for productive viral entry.

Interdomain Stabilization Impairs CD4 Binding and Improves Immunogenicity of the HIV-1 Envelope Trimer.

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines.

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Structure of the transmembrane domain of HIV-1 envelope glycoprotein.

HIV-1 envelope spike (Env) is a heavily glycosylated, type I membrane protein that mediates fusion of viral and cell membranes to initiate infection. It is also a primary target of neutralizing antibodies and thus an important candidate for vaccine development. We have recently reported a nuclear magnetic resonance structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in a membrane-like environment. Taking HIV-1 as an example, we discuss here how a TM domain can anchor, stabilize, and modulate a viral envelope spike and how its high-resolution structure can contribute to understanding viral membrane fusion and to immunogen design.

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins.

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Effect of Several HIV Antigens Simultaneously Loaded with G2-NN16 Carbosilane Dendrimer in the Cell Uptake and Functionality of Human Dendritic Cells.

Dendrimers are highly branched, star-shaped, and nanosized polymers that have been proposed as new carriers for specific HIV-1 peptides. Dendritic cells (DCs) are the most-potent antigen-presenting cells that play a major role in the development of cell-mediated immunotherapy due to the generation and regulation of adaptive immune responses against HIV-1. This article reports on the associated behavior of two or three HIV-derived peptides simultaneously (p24/gp160 or p24/gp160/NEF) with cationic carbosilane dendrimer G2-NN16. We have found that (i) immature DCs (iDCs) and mature (mDCs) did not capture efficiently HIV peptides regarding the uptake level when cells were treated with G2-NN16-peptide complex alone; (ii) the ability of DCs to migrate was not depending on the peptides presence; and (iii) with the use of molecular dynamic simulation, a mixture of peptides decreased the cell uptake of the other peptides (in particular, NEF hinders the binding of more peptides and is especially obstructing of the binding of gp160 to G2-NN16). The results suggest that G2-NN16 cannot be considered as an alternative carrier for delivering two or more HIV-derived peptides to DCs.

HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption.

Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117,a broad and potent neutralizing antibody against the CD4 binding site of the HIV-1 Env protein, during analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Results show that two or four 30 mg kg(-1) 3BNC117 infusions,separated by 3 or 2 weeks, respectively, are generally well tolerated.Infusions are associated with a delay in viral rebound of 5-9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks, respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals,emerging viruses show increased resistance, indicating escape.However, 30% of participants remained suppressed until antibody concentrations waned below 20 µg ml(-1), and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks.We conclude that the administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.

Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 µg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1.

Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine.

Discrete partitioning of HIV-1 Env forms revealed by viral capture.

BACKGROUND: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env. RESULTS: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps. CONCLUSIONS: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions.

In vitro assembly of a viral envelope.

Viruses such as influenza and Ebola are enveloped in lipid bilayers annexed from host cells and containing glycoproteins essential for the infection process. At the molecular level little is known about the assembly process in terms of physical interactions between the lipids and glycoproteins. In this paper we assemble HIV glycoproteins in lipid vesicles in order to examine envelope assembly, a process that is usually only executed under control of a host cell. Using atomic force microscopy it was possible to observe fusion of individual envelope like particles, and contrast this with the behaviour of lipid vesicles without envelope glycoproteins. It was found that the inclusion of glycoproteins caused the vesicles to distort and that the subsequent fusion "footprint" with a lipid bilayer was related to the envelopes' unique morphology. This non-spherical morphology suggests that the presence of a viral capsid may be essential for the stability of an enveloped virus. Interactions between trans-membrane gp41 and gp120, the spikes protruding from a virion, were examined using supported lipid bilayers. Interactions between the gp120 and membrane-located gp41 resulted in the assembly of unusual molecular wires, one molecule in height and with a zigzag arrangement of gp120 molecules. In this work we have shown that purely physical/chemical interactions have dramatic effects on glycoprotein/lipid assembly and should be considered in the development of virus based technologies such as virosomes.

HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein.

A major goal for HIV-1 vaccine development is the production of an immunogen to mimic native, functional HIV-1 envelope trimeric spikes (Env) on the virion surface. We lack a reliable description of a native, functional trimer, however, because of inherent instability and heterogeneity in most preparations. We describe here two conformationally homogeneous Envs derived from difficult-to-neutralize primary isolates. All their non-neutralizing epitopes are fully concealed and independent of their proteolytic processing. Most broadly neutralizing antibodies (bnAbs) recognize these native trimers. Truncation of their cytoplasmic tail has little effect on membrane fusion, but it diminishes binding to trimer-specific bnAbs while exposing non-neutralizing epitopes. These results yield a more accurate antigenic picture than hitherto possible of a genuinely untriggered and functional HIV-1 Env; they can guide effective vaccine development.

Honing a harder-hitting hammerhead improves broadly neutralizing antibody breadth and potency.

While current HIV-1 therapies have greatly improved the quality and duration of life for infected individuals, a vaccine to prevent transmission of the virus is lacking. Broadly neutralizing monoclonal antibodies (bnmAbs) with the capacity to neutralize multiple HIV-1 variants have been isolated from HIV-1-infected individuals, and there has been a great effort to investigate how these bnmAbs arise, due their potential for HIV-1 vaccination. In this issue of the JCI, Willis and colleagues apply a computational approach to design variants of the bnmAb PG9 in an attempt to enhance potency and neutralization breadth. One of these variants was able to target multiple PG9-resistant strains, as the result of stabilization of the long heavy chain complementarity determining region 3 (HCDR3). The results of this study provide important insight and a unique approach to optimizing HIV-1 bnmABs.

Redesigned HIV antibodies exhibit enhanced neutralizing potency and breadth.

Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100(F)Y, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100(F)Y fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100(F)Y, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies.

Identifiable biomarker and treatment development using HIV-1 long term non-progressor sera.

BACKGROUND: HIV-infected long-term non-progressor (LTNP) subjects can prevent viral replication and may harbor useful information for the development of both antibody and active vaccination treatments. In this study we used LTNP sera to examine the epitopes presented to the gp160 protein, and from this procedure we hope to elucidate potential biomarkers pertaining to the level of resistance a patient may have in developing AIDS after infection with HIV. We used five clinical sera samples from LTNP patients to identify common epitopes by ELISA; peptides with high binding to sera were selected and analyzed for conservation among HIV clades. Antibodies were generated against one identified epitope using a chimeric peptide in BALB/c mice, and both the sera from these mice and LTNP sera were tested for viral inhibition capabilities. RESULTS: A monoclonal antibody, CL3, against one identified epitope was used to compare these epitopes neutralizing capability. LTNP sera was also studied to determine chemokine/cytokine changes in these patients. The sera from LTNP patients 2, 3, 4, and 5 were identified as having the highest titers, and also significantly inhibited syncytia formation in vitro. Finally, the protein cytokine array demonstrated that I-309 and IGFBP-1 decreased in LTNPs, but levels of TIMP-1 and NAP-2 increased significantly. CONCLUSIONS: Our results indicate that the use of LTNP samples may be a useful for identifying further anti-viral epitopes, and may be a possible predictor for determining if patients show higher resistances of converting the HIV infection to AIDS.