Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics ...of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host ...biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.
HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man
-V3") for structural studies of an HIV Env ...third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man
-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.
Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To ...determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans—a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.
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•Env vaccination elicits antibodies that target the V3-glycan-neutralizing epitope•Repetitive vaccination with a single Env over 4 years induced V3-glycan antibodies•V3-glycan bnAb precursors recognize Env Man9GlcNAc2 to neutralize
Most bnAb epitopes on HIV-1 Envelope include host glycans, but previous Env vaccines have not induced glycan-dependent antibodies. Saunders et al. describe here the ontogeny, crystal structure with glycan, and virion Man9GlcNAc2-dependent neutralization for glycan-reactive antibodies induced by envelope vaccination.
A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) ...third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man
-V3). V3-glycan bnAbs bound to Man
-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man
-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1-infected individual. In rhesus macaques, immunization with Man
-V3 induced V3-glycan-targeted antibodies. Thus, the Man
-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.
Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of ...prophylactic and therapeutic vaccines. However, clinical use of QS21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure-activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in
adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the
saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants.
We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type-specific labeling using amino acid precursors (CTAP). Through ...transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope-labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers.
Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of ...prophylactic and therapeutic vaccines. However, clinical use of QS-21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure-activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in
in vivo
adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the
Quillaja
saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants.
A mouse vaccination model and molecular dynamics studies reveal characteristic conformations of active QS-21 variants.
A mouse vaccination model and molecular dynamics studies reveal characteristic conformations of active QS-21 variants.
Immunological adjuvants such as the saponin natural product QS-21 help stimulate ...the immune response to co-administered antigens and have become increasingly important in the development of prophylactic and therapeutic vaccines. However, clinical use of QS-21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure–activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in
in vivo
adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the
Quillaja
saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants.
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