Alberto Fernández-Tejada

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

Significance A current key goal of HIV-1 vaccine development is to learn how to induce antibodies that will neutralize many diverse HIV-1 strains. Current HIV-1 vaccines elicit strain-specific neutralizing antibodies, whereas broadly neutralizing antibodies (BnAbs) are not induced and only arise in select HIV-1 chronically infected individuals. One strategy for induction of favored antibody responses is to design and produce homogeneous immunogens with selective expression of BnAb but not dominant epitopes. In this study, we describe the binding properties of chemically synthesized variable loop 1/2 (V1V2) glycopeptides that bind both to mature HIV-1 envelope BnAbs and the receptors of their naïve B cells. These results demonstrate that such synthetic glycopeptides can be immunogens that selectively target BnAb naïve B cells. Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1–infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.

Chemical synthesis of highly congested gp120 V1V2 N-glycopeptide antigens for potential HIV-1-directed vaccines.

Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn(160) and Asn(156). Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.

Synthesis and Preclinical Evaluation of QS-21 Variants Leading to Simplified Vaccine Adjuvants and Mechanistic Probes

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

Development of a minimal saponin vaccine adjuvant based on QS-21

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability, and an enigmatic molecular mechanism of action. Herein, we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained at the injection site and nearest draining lymph nodes preferentially compared to attenuated variants. Overall, these studies have yielded critical insights into saponin structure–function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

Insights into the Geometrical Features Underlying β‐O‐GlcNAc Glycosylation: Water Pockets Drastically Modulate the Interactions between the Carbohydrate and the Peptide Backbone

There is an increasing interest in clarifying the role of bN-acetyl-d-glucosamine (b-GlcNAc) since the discovery that it modifies the chemical, biochemical, and biomedical behavior of an increasing number of cytoplasmic and nuclear proteins, such as transcription factors, nuclear pore proteins, oncogene products, tumor suppressors, and cytoskeletal proteins. This post-translational glycosylation is highly dynamic and draws comparisons with protein phosphorylation as a biological control mechanism. It has been implicated in gene transcription, nuclear trafficking, protein translation, signal transduction, regulation of protein–protein interactions, and the sensing of nutritional levels within the cell. Furthermore, there is clear evidence that the aberrant OGlcNAc modification of proteins is correlated with diabetes, tumorgenesis, and even with Alzheimer s disease. From a structural point of view, O-GlcNAc glycosylation sites do not show obvious consensus sequences. Additionally, in contrast to classical protein glycosylation, O-GlcNAc is not elongated or further modified with a complex array of glycans. However, in spite of the importance of O-GlcNAc glycosylation, sparse information has been reported to date concerning the geometry and dynamics of b-O-GlcNAc-Ser and b-O-GlcNAc-Thr motifs. On the contrary, significant progress has been made with regard to the understanding of the structural properties of the b-N-GlcNAc-Asn fragment. In fact, while numerous crystal structures containing this fragment have been reported to date, only one crystal structure with a serine residue glycosylated with a b-O-GlcNAc has been deposited in the Protein Data Bank. This crystal structure shows the complex between a b-O-GlcNAc glycopeptide with the sequence FAPSNYPAL (named K3G) and the MHC Class 1 H-2D antibody. The authors suggested the occurrence of two conformations for the O-GlcNAc-Ser glycosidic linkage, each displaying similar occupancy. This experimental finding clearly suggests a significant degree of mobility for this moiety. One of these conformers is shown in Figure 1. On the other hand, the conformational study in aqueous solution of glycopeptides containing the b-O-GlcNAc-Ser/

Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide

A synthetic glycopeptide mimics a key neutralizing epitope on the HIV-1 envelope and can be used to isolate HIV-1 broadly neutralizing antibodies. Guiding anti-glycan antibodies Although it typically evades the immune system, HIV does have sites of vulnerability that can be targeted in vaccine design. One such site is a glycan near the V3 loop of the envelope protein, but antibodies recognizing this epitope are often not detected in people infected with HIV. Alam et al. designed a synthetic glycopeptide that can identify B cells targeting this epitope and also used it to immunize macaques. Bonsignori et al. used this synthetic glycopeptide and other baits to study the V3-glycan antibody responses of an HIV-infected individual that developed broadly neutralizing antibodies. They also examined viral evolution over time and found clues as to why these types of antibodies do not develop more often. These tools and findings could pave the way for a vaccine that protects against diverse strains of HIV. 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 (Man9-V3). V3-glycan bnAbs bound to Man9-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man9-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 Man9-V3 induced V3-glycan-targeted antibodies. Thus, the Man9-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.

Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique β-subunit, that form noncovalent heterodimers. Structurally, β-hCG shares a high degree of sequence similarity with β-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous β-hLH and β-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of β-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (β-hCG) as well as its closely related homologue (β-hLH) as discrete glycoforms.

Development of Improved Vaccine Adjuvants Based on the Saponin Natural Product QS-21 through Chemical Synthesis

Vaccines based on molecular subunit antigens are increasingly being investigated due to their improved safety and more precise targeting compared to classical whole-pathogen vaccines. However, subunit vaccines are inherently less immunogenic; thus, coadministration of an adjuvant to increase the immunogenicity of the antigen is often necessary to elicit a potent immune response. QS-21, an immunostimulatory saponin natural product, has been used as an adjuvant in conjunction with various vaccines in numerous clinical trials, but suffers from several inherent liabilities, including scarcity, chemical instability, and dose-limiting toxicity. Moreover, little is known about its mechanism of action. Over a decade-long effort, beginning at the University of Illinois at Urbana-Champaign and continuing at the Memorial Sloan Kettering Cancer Center (MSKCC), the group of Prof. David Y. Gin accomplished the total synthesis of QS-21 and developed a practical semisynthetic approach to novel variants that overcome the liabilities of the natural product. First, semisynthetic QS-21 variants were designed with stable amide linkages in the acyl chain domain that exhibited comparable in vivo adjuvant activity and lower toxicity than the natural product. Further modifications in the acyl chain domain and truncation of the linear tetrasaccharide domain led to identification of a trisaccharide variant with a simple carboxylic acid side chain that retained potent adjuvant activity, albeit with reemergence of toxicity. Conversely, an acyl chain analogue terminating in a free amine was inactive but enabled chemoselective functionalization with radiolabeled and fluorescent tags, yielding adjuvant-active saponin probes that, unlike inactive congeners, accumulated in the lymph nodes in vaccinated mice and internalized into dendritic cells. Subtle variations in length, stereochemistry, and conformational flexibility around the central glycosidic linkage provided QS-21 variants with adjuvant activities that correlated with specific conformations found in molecular dynamics simulations. Notably, deletion of the entire branched trisaccharide domain afforded potent, truncated saponin variants with negligible toxicity and improved synthetic access, facilitating subsequent investigation of the triterpene core. The triterpene C4-aldehyde substituent, previously proposed to be important for QS-21 adjuvant activity, proved to be dispensable in these truncated saponin variants, while the presence of the C16 hydroxyl group enhanced activity. Novel adjuvant conjugates incorporating the small-molecule immunopotentiator tucaresol at the acyl chain terminus afforded adjuvant-active variants but without significant synergistic enhancement of activity. Finally, a new divergent synthetic approach was developed to provide versatile and streamlined access to additional linear oligosaccharide domain variants with modified sugars and regiochemistries, opening the door to the rapid generation of diverse, synthetically accessible analogues. In this Account, we summarize these multidisciplinary studies at the interface of chemistry, immunology, and medicine, which have provided critical information on the structure-activity relationships (SAR) of this Quillaja saponin class; access to novel, potent, nontoxic adjuvants for use in subunit vaccines; and a powerful platform for investigations into the mechanisms of saponin immunopotentiation.

Designing synthetic vaccines for HIV

Despite three decades of intensive research efforts, the development of an effective prophylactic vaccine against HIV remains an unrealized goal in the global campaign to contain the HIV/AIDS pandemic. Recent characterization of novel epitopes for inducing broadly neutralizing antibodies has fueled research in the design and synthesis of new, well-defined antigenic constructs for the development of HIV envelope-directed vaccines. The present review will cover previous and recent efforts toward the design of synthetic vaccines based on the HIV viral envelope glycoproteins, with special emphasis on examples from our own laboratories. The biological evaluation of some of the most representative vaccine candidates, in terms of their antigenicity and immunogenicity, will also be discussed to illustrate the current state-of-the-art toward the development of fully synthetic HIV vaccines.

Design, synthesis, and immunologic evaluation of vaccine adjuvant conjugates based on QS-21 and tucaresol

Immunoadjuvants are used to potentiate the activity of modern subunit vaccines that are based on molecular antigens. An emerging approach involves the combination of multiple adjuvants in a single formulation to achieve optimal vaccine efficacy. Herein, to investigate such potential synergies, we synthesized novel adjuvant conjugates based on the saponin natural product QS-21 and the aldehyde tucaresol via chemoselective acylation of an amine at the terminus of the acyl chain domain in QS saponin variants. In a preclinical mouse vaccination model, these QS saponin-tucaresol conjugates induced antibody responses similar to or slightly higher than those generated with related QS saponin variants lacking the tucaresol motif. The conjugates retained potent adjuvant activity, low toxicity, and improved activity-toxicity profiles relative to QS-21 itself and induced IgG subclass profiles similar to those of QS-21, indicative of both Th1 cellular and Th2 humoral immune responses. This study opens the door to installation of other substituents at the terminus of the acyl chain domain to develop additional QS saponin conjugates with desirable immunologic properties.