ABSTRACT
Broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected donors are vaccine paradigms. These bNAbs recognize envelope glycoprotein trimers that carry 75-90 oligomannose and complex-type glycans. Although bNAbs and their precursors must navigate past glycans, they usually also make some glycan contacts. Glycan-modified vaccines may therefore be useful to initiate and guide bNAb development. Here, we describe two ways to modify Env glycans for possible vaccine use: 1) using a cocktail of glycosidases (termed "NGAF3" (Neuraminidase, ß-Galactosidase, N-Acetylglucosaminidase, endoglycosidase F3 (endo F3)) to deplete complex glycans to try to minimize bNAb-glycan clashes and 2) co-expressing ß-1,4-galactosyltransferase 1 (B4G) and ß-galactoside α-2,6 sialyltransferase 1 (ST6) during Env biosynthesis, creating bNAb-preferred glycan structures. Mass spectrometry revealed that NGAF3 removed glycan heads at 3/7 sites occupied by complex glycans. B4G overexpression resulted in hybrid glycan development whenever complex glycans were closely spaced. The glycan at position 611 in of Env's gp41 transmembrane subunit was uniquely isolated from the effects of both endo F3 and B4G. B4G and ST6 co-expression increased hybrid and sialylated glycan abundance, reducing glycan complexity. In rabbit vaccinations, B4G + ST6 virus-like particles (VLPs) induced less frequent, weaker titer NAbs, implying that ST6-mediated increased Env charge dampens vaccine antibodies. In some cases, vaccine sera preferentially neutralized B4G + ST6-modified pseudovirus. HIV-1+ donor plasma NAbs were generally more effective against B4G + ST6 modified pseudovirus, suggesting a preference for less complex and/or α-2,6 sialylated Env trimers. Collectively, our data suggest that B4G and ST6 Env modifications are best suited for intermediate or late vaccine shots.
ABSTRACT
Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.
Subject(s)
HIV Envelope Protein gp41 , HIV-1 , Humans , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , Glycosylation , Mannose/metabolism , Mutation , Glycoproteins/metabolism , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV AntibodiesABSTRACT
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.
Subject(s)
HIV Infections , HIV-1 , Antibodies, Neutralizing , Binding Sites , Glycoproteins , HIV Antibodies , HIV Envelope Protein gp120/genetics , HIV-1/genetics , Humans , Malaysia , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
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.
Subject(s)
AIDS Vaccines/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , Broadly Neutralizing Antibodies/immunology , Epitopes/immunology , HIV Antibodies/immunology , HumansABSTRACT
Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the â¼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.
Subject(s)
HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp160/metabolism , HIV-1/metabolism , Protein Processing, Post-Translational , Cysteine , HEK293 Cells , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp160/genetics , HIV-1/genetics , HIV-1/growth & development , HeLa Cells , Humans , Protein Folding , Protein Interaction Domains and Motifs , Protein Sorting Signals , Protein Stability , Structure-Activity Relationship , Viral Load , Virus ReplicationABSTRACT
The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing ß-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE-thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , Antibodies, Neutralizing/blood , Epitopes/immunology , Glycosylation , HIV Antibodies/immunology , HIV Envelope Protein gp120/blood , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/blood , HIV Envelope Protein gp41/metabolism , HIV Infections/immunology , HIV Infections/therapy , Humans , Leukocytes, Mononuclear/immunology , Polysaccharides/immunology , Polysaccharides/metabolism , Protein ConformationABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV-1/immunology , Peptide Fragments/immunology , Protein Conformation , env Gene Products, Human Immunodeficiency Virus/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Binding Sites , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/immunology , HIV Antibodies/chemistry , HIV Antibodies/metabolism , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV Infections/virology , Humans , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phylogeny , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Vaccines, Virus-Like Particle/chemistry , Vaccines, Virus-Like Particle/immunology , Vaccines, Virus-Like Particle/metabolismABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/immunology , CD4 Antigens/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , Animals , Antibodies, Neutralizing/biosynthesis , Binding Sites/genetics , Binding Sites/immunology , Cell Line , Epitopes/genetics , Epitopes/immunology , Female , HEK293 Cells , HIV Antibodies/biosynthesis , Humans , Immunization , Neutralization Tests , RabbitsABSTRACT
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.
Subject(s)
Cell-Derived Microparticles/metabolism , Diabetes Mellitus, Type 1/immunology , Endogenous Retroviruses/immunology , Gene Products, env/metabolism , Gene Products, gag/metabolism , Islets of Langerhans/immunology , Mesenchymal Stem Cells/metabolism , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Autoantibodies/blood , Autoimmunity , Cell-Derived Microparticles/immunology , Cells, Cultured , Female , Gene Products, env/genetics , Gene Products, gag/genetics , Humans , Islets of Langerhans/metabolism , Lymphocyte Activation , Mesenchymal Stem Cells/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , RNA, Small Interfering/genetics , T-Lymphocytes/transplantationABSTRACT
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.
Subject(s)
AIDS Vaccines/immunology , Epitope Mapping/methods , Epitopes/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , Algorithms , Antibody Formation , Antibody Specificity , Cohort Studies , Computer Simulation , HIV Infections/virology , Humans , Neutralization TestsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/physiology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , CD4 Antigens/immunology , Crystallography, X-Ray , Epitopes/immunology , HEK293 Cells , HIV Infections/virology , HIV-1/chemistry , HIV-1/immunology , Humans , Models, Molecular , Protein Conformation , Protein Multimerization , Virus InternalizationABSTRACT
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.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , CD4 Antigens/metabolism , Epitopes/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/metabolism , HIV-1/immunology , Polysaccharides/deficiency , Animals , Binding Sites , CD4 Antigens/genetics , Epitopes/chemistry , Female , Guinea Pigs , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV Infections/virology , Humans , Immunization , Polysaccharides/chemistry , Polysaccharides/genetics , Protein Conformation , RabbitsABSTRACT
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) <50 µg ml(-1). The median IC50 of neutralized viruses was 0.033 µg ml(-1), among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.
Subject(s)
Antibodies, Neutralizing/immunology , Antibody Affinity , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/pharmacology , Antibody Specificity , CD4 Antigens/metabolism , Cell Line , Cell Membrane/virology , Conserved Sequence , Epitope Mapping , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/chemistry , HIV Antibodies/genetics , HIV Antibodies/pharmacology , HIV-1/drug effects , HIV-1/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/ultrastructure , Inhibitory Concentration 50 , Leukocytes, Mononuclear , Models, Molecular , Molecular Sequence Data , Receptors, CCR5/metabolism , Virus Internalization/drug effectsABSTRACT
Virus-like particles (VLPs) offer a platform to test the hypothesis that, since antibody binding to native envelope glycoprotein (Env) trimers results in HIV-1 neutralization, that native Env trimers presented in membranes may be useful for inducing neutralizing antibodies (nAbs) in a vaccine setting. So far, VLPs have not fulfilled this potential. Here, using a "shotgun" approach, we evaluated a wide cross-section of variables in a series of VLP immunizations. We identified 3 tentative leads. First, that VLP doses may not have been sufficient for optimal nAb induction. Second, that dampening the antigenicity of non-functional Env (for example uncleaved gp160) using either protease digests or IgG masking may be useful. Third, that guinea pig sera preferentially target non-conserved epitopes and exhibit relatively high background activity, suggesting that rabbits may be preferable as small animal vaccine models. Recent immunogenicity studies in rabbits appear to bear out all 3 of these leads.
ABSTRACT
Virus-like particles (VLPs) offer a platform to test the hypothesis that, since antibody binding to native envelope glycoprotein (Env) trimers results in HIV-1 neutralization, that native Env trimers presented in membranes may be useful for inducing neutralizing antibodies (nAbs) in a vaccine setting. So far, VLPs have not fulfilled this potential. Here, using a "shotgun" approach, we evaluated a wide cross-section of variables in a series of VLP immunizations. We identified 3 tentative leads. First, that VLP doses may not have been sufficient for optimal nAb induction. Second, that dampening the antigenicity of non-functional Env (for example uncleaved gp160) using either protease digests or IgG masking may be useful. Third, that guinea pig sera preferentially target non-conserved epitopes and exhibit relatively high background activity, suggesting that rabbits may be preferable as small animal vaccine models. Recent immunogenicity studies in rabbits appear to bear out all 3 of these leads.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/blood , HIV Antibodies/blood , HIV-1/immunology , Vaccines, Virus-Like Particle/immunology , Animals , Female , Guinea Pigs , Macaca mulatta , Male , Models, Animal , RabbitsABSTRACT
Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp160/chemistry , HIV Envelope Protein gp160/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/isolation & purification , Antibody Affinity/genetics , Antibody Affinity/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Binding Sites/immunology , CD4 Antigens/immunology , CD4 Antigens/metabolism , Cell Lineage , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Epitope Mapping , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Evolution, Molecular , HIV Antibodies/chemistry , HIV Antibodies/genetics , HIV Antibodies/isolation & purification , HIV Infections/immunology , HIV-1/chemistry , HIV-1/immunology , Humans , Models, Molecular , Molecular Sequence Data , Neutralization Tests , Protein Structure, Tertiary , Somatic Hypermutation, Immunoglobulin/geneticsABSTRACT
Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibody Specificity/immunology , Binding Sites, Antibody/genetics , Binding Sites, Antibody/immunology , CD4 Antigens/immunology , CD4 Antigens/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Glycosylation , HIV Antibodies/metabolism , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV-1/classification , HIV-1/metabolism , Humans , Molecular Sequence Data , Mutation , Neutralization Tests , Protein Binding/immunology , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant ΔV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the ΔV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.
Subject(s)
AIDS Vaccines/administration & dosage , Antibodies, Neutralizing/immunology , Antibody Formation/immunology , HIV Antibodies/immunology , HIV Infections/prevention & control , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/immunology , Animals , Antibodies, Neutralizing/blood , Epitopes/immunology , HIV Antibodies/blood , HIV Infections/immunology , HIV Infections/virology , Humans , Neutralization Tests , Rabbits , Sequence Deletion , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
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.