Glycopeptide Conjugates Research Articles

Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope

Recently, we reported a directed evolution method which enabled us to discover sequences of glycopeptides that bind with picomolar affinity to HIV antibody 2G12 and are of interest as HIV vaccine candidates. In this manuscript, we describe the syntheses of several of these large (∼11–12 kDa) glycopeptides by a combination of fast flow peptide synthesis and click chemistry. We also discuss the optimization of their attachment to carrier protein CRM197, affording antigenic and immunogenic conjugates ready for animal vaccination.

Synthesis and immunological evaluation of MUC1 glycopeptide conjugates bearing N-acetyl modified STn derivatives as anticancer vaccines.

Glycoprotein MUC1 is an attractive target for anti-tumor vaccine development. However, the weak immunogenicity of MUC1 remains a significant problem. To solve this problem, several STn derivatives with N-acetyl modifications were synthesized and incorporated into a 20-amino acid MUC1 tandem repeat sequence. The modified STn-MUC1 glycopeptides were further connected to a carrier protein keyhole limpet hemocyanin (KLH). The immunological effects of these synthetic vaccine conjugates were evaluated using the BALB/c mouse model. The results showed that vaccine V2 elicited higher titers of antibodies which cross-reacted with the native STn-MUC1 antigen. Moreover, the elicited antisera reacted with the STn-MUC1 antigen-positive tumor cells, indicating that the carbohydrate antigen modification strategy may hold potential to overcome the weak immunogenicity of natural MUC1 glycopeptides.

Synthesis and biological evaluation of a novel MUC1 glycopeptide conjugate vaccine candidate comprising a 4'-deoxy-4'-fluoro-Thomsen-Friedenreich epitope.

The development of selective anticancer vaccines that provide enhanced protection against tumor recurrence and metastasis has been the subject of intense research in the scientific community. The tumor-associated glycoprotein MUC1 represents a well-established target for cancer immunotherapy and has been used for the construction of various synthetic vaccine candidates. However, many of these vaccine prototypes suffer from an inherent low immunogenicity and are susceptible to rapid in vivo degradation. To overcome these drawbacks, novel fluorinated MUC1 glycopeptide-BSA/TTox conjugate vaccines have been prepared. Immunization of mice with the 4’F-TF-MUC1-TTox conjugate resulted in strong immune responses overriding the natural tolerance against MUC1 and producing selective IgG antibodies that are cross-reactive with native MUC1 epitopes on MCF-7 human cancer cells.

The development of synthetic antitumour vaccines from mucin glycopeptide antigens

Based on important cell-biological and biochemical results concerning the structural difference between membrane glycoproteins of normal epithelial cells and epithelial tumour cells, tumour-associated glycopeptide antigens have been chemically synthesised and structurally confirmed. Glycopeptide structures of the tandem repeat sequence of mucin MUC1 of epithelial tumour cells constitute the most promising tumour-associated antigens. In order to generate a sufficient immunogenicity of these endogenous structures, usually tolerated by the immune system, these synthetic glycopeptide antigens were conjugated to immune stimulating components: in fully synthetic two-component vaccines either with T-cell peptide epitopes or with Toll-like receptor2 lipopeptide ligands or in three-component vaccines with both these stimulants. Alternatively, the synthetic glycopeptide antigens were coupled to immune stimulating carrier proteins. In particular, MUC1 glycopeptide conjugates with Tetanus toxoid proved to be efficient vaccines inducing very strong immune responses in mice. The antibodies elicited with the fully synthetic vaccines showed selective recognition of the tumour-associated glycopeptides as was shown by neutralisation and micro-array binding experiments. After booster immunisations, most of the immune responses showed the installation of an immunological memory. Immunisation with fully synthetic three-component vaccines induced immune reactions with therapeutic effects in terms of reduction of the tumour burden in mice or in killing of tumour cells in culture, while MUC1 glycopeptide-Tetanus toxoid vaccines elicited antibodies in mice which recognised tumour cells in human tumour tissues. The results achieved so far are considered to be promising for the development of an active immunisation against tumours.

Self-Adjuvanting Glycopeptide Conjugate Vaccine against Disseminated Candidiasis

Our research on pathogenesis of disseminated candidiasis led to the discovery that antibodies specific for Candida albicans cell surface β-1, 2–mannotriose [β-(Man)3] protect mice. A 14 mer peptide Fba, which derived from the N-terminal portion of the C. albicans cytosolic/cell surface protein fructose-bisphosphate aldolase, was used as the glycan carrier and resulted in a novel synthetic glycopeptide vaccine β-(Man)3-Fba. By a dendritic cell-based immunization approach, this conjugate induced protective antibody responses against both the glycan and peptide parts of the vaccine. In this report, we modified the β-(Man)3-Fba conjugate by coupling it to tetanus toxoid (TT) in order to improve immunogenicity and allow for use of an adjuvant suitable for human use. By new immunization procedures entirely compatible with human use, the modified β-(Man)3-Fba-TT was administered either alone or as a mixture made with alum or monophosphoryl lipid A (MPL) adjuvants and given to mice by a subcutaneous (s.c.) route. Mice vaccinated with or, surprisingly, without adjuvant responded well by making robust antibody responses. The immunized groups showed a high degree of protection against a lethal challenge with C. albicans as evidenced by increased survival times and reduced kidney fungal burden as compared to control groups that received only adjuvant or DPBS buffer prior to challenge. To confirm that induced antibodies were protective, sera from mice immunized against the β-(Man)3-Fba-TT conjugate transferred protection against disseminated candidiasis to naïve mice, whereas C. albicans-absorbed immune sera did not. Similar antibody responses and protection induced by the β-(Man)3-Fba-TT vaccine was observed in inbred BALB/c and outbred Swiss Webster mice. We conclude that addition of TT to the glycopeptide conjugate results in a self-adjuvanting vaccine that promotes robust antibody responses without the need for additional adjuvant, which is novel and represents a major step forward in vaccine design against disseminated candidiasis.

A sweet cleft in HIV's armour

The structure of an antibody that potently neutralizes a wide range of HIV-1 strains, together with a minimal antigen mimic, is an advance towards the design of vaccines that may elicit protective responses. See Article p.336 The V1/V2 variable region of the gp120 envelope glycoprotein of HIV-1, with its extraordinary sequence diversity and N-linked glycosylation, exemplifies the ability of the virus to evade antibody recognition. It has also resisted structural determination. Now Peter Kwong and colleagues have determined the atomic-level structure of gp120 V1/V2 by using an antibody called PG9, which can neutralize most strains of HIV. Instead of being confounded by the N-linked glycan that shields most of gp120 from immune recognition, PG9 uses N-linked glycan for binding through a mechanism shared by a number of antibodies capable of effective HIV neutralization. The structure shows that the antibody recognizes glycopeptide conjugates and avoids diversity in V1/V2 by making sequence-independent interactions, such as hydrogen bonds.

Vaccine and Monoclonal Antibody That Enhance Mouse Resistance to Candidiasis

Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)(3)] on the cell surface of Candida albicans protect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526-13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion of C. albicans wall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived from fructose-bisphosphate aldolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by different C. albicans strains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease.

Towards a Fully Synthetic MUC1‐Based Anticancer Vaccine: Efficient Conjugation of Glycopeptides with Mono‐, Di‐, and Tetravalent Lipopeptides Using Click Chemistry

The membrane-bound tumor-associated glycoprotein MUC1 is aberrantly glycosylated in cancer cells compared with normal cells, and is therefore considered an attractive target for cancer immunotherapy. However, tumor-associated glycopeptides from MUC1 do not elicit a sufficiently robust immune response. Therefore, antitumor vaccines were developed, which consist of MUC1 glycopeptides as the B epitopes and immune-stimulating toll-like receptor 2 (TLR 2) lipopeptide ligands. These fully synthetic vaccine candidates were prepared by solid-phase synthesis of the MUC1 glycopeptides. The Pam(3) Cys lipopeptide, also synthesized on solid-phase, was C-terminally coupled to oligovalent lysine cores, which N-terminally incorporate O-propargyl oligoethylene glycol acyl side chains. The MUC1 glycopeptides and lipopeptide lysine constructs were then conjugated by click chemistry to give oligovalent synthetic vaccines. Oligovalent glycopeptide-lipopeptide conjugates are considered more immunogenic than their monovalent analogues.

N-Glycan Targeted Gene Delivery to the Dendritic Cell SIGN Receptor

A novel nonviral gene delivery vector composed of a high-mannose N-glycan conjugated to a polyacridine peptide was prepared. The glycopeptide was designed to bind to plasmid DNA by a combination of polyintercalation and ionic binding, and to the DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin) receptor expressed on CHO cells by recognition of the high-mannose N-glycan. The glycopeptide conjugate was prepared by purification of a high-mannose N-glycan from affinity fractionated soybean agglutinin (SBA). The SBA was proteolyzed to release the N-glycan which was then modified on its N-terminus with Tyr and a propionate maleimide. A DNA binding polyacridine peptide, Cys-(Acr-Lys)(4), was prepared by solid-phase peptide synthesis using Fmoc-Lys(Acr), then conjugated to the maleimide on the N-glycan to produce a glycopeptide. The glycopeptide bound to DNA with high affinity as determined by fluorophore displacement assay and DNA band shift on agarose gel. When bound to Cy5 labeled DNA, the glycopeptide mediated specific uptake in DC-SIGN CHO (+) cells as determined by FACS analysis. In vitro gene transfer studies established that the glycopeptide increased the specificity of gene transfer in DC-SIGN CHO (+) cells 100-fold relative to CHO (-) cells. These studies suggest that a high-mannose N-glycan conjugated to a polyacridine peptide may also facilitate receptor mediated gene delivery in dendritic cells and thereby find utility in the delivery of DNA vaccines.

Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

The first fully synthetic glycopeptide vaccines against a fungal disease have been used to combat disseminated candidiasis in mice. Six T cell peptides found in Candida albicans cell wall proteins were selected by algorithm peptide epitope searches; each was synthesized and conjugated to the fungal cell wall beta-mannan trisaccharide [beta-(Man)(3)] by novel saccharide-peptide linker chemistry to create glycopeptide conjugates. The six proteins were selected because of expression during human candidiasis and cell wall association and included: fructose-bisphosphate aldolase (Fba); methyltetrahydropteroyltriglutamate (Met6); hyphal wall protein-1 (Hwp1); enolase (Enol); glyceraldehyde-3-phosphate dehydrogenase (Gap1); and phosphoglycerate kinase (Pgk1). By immunization protocols favoring production of protective antibody, the beta-(Man)(3)-Fba, beta-(Man)(3)-Met6 and beta-(Man)(3)-Hwp1 induced protection evidenced by survival and reduced kidney fungal burden, the beta-(Man)(3)-Eno1 and beta-(Man)(3)-Gap1 gave moderate protection, and the beta-(Man)(3)-Pgk1 slightly enhanced disease. For the beta-(Man)(3)-Fba conjugate, protection was uniquely acquired through immunity against the carbohydrate and the Fba peptide. This approach based on fully synthetic chemically defined immunogens should be generally useful in vaccine development.

Glycoproteomics Using Chemical Immobilization

Protein glycosylation is prevalent in proteins destined for extracellular environments, e.g., transmembrane proteins, cell surface proteins, and secreted proteins in tissues and body fluids. These also are the proteins that are most easily accessible for diagnostic and therapeutic purposes. This unit describes methods for solid-phase extraction of glycopeptides and subsequent identification of glycopeptides as well as glycosylation sites. The extraction is based on the conjugation of glycopeptides to a solid support, using hydrazide chemistry, and the specific release of formerly glycosylated peptides. The recovered peptides are then identified by tandem mass spectrometry. The methods are applied to the analysis of proteins from cells, body fluids, and tissues.

Spacer-separated sialyl LewisX cyclopeptide conjugates as potential E-selectin ligands

Completely protected sialyl LewisX azide was synthesized from a neolactosamine azide precursor carrying a 3- O-allyloxycarbonyl group as the temporary protecting group. After its Pd(0)-catalyzed deprotection and stereoselective α-fucosylation, the obtained LewisX azide was subjected to O-deacetylation in the galactose unit and subsequent regio- and stereoselective sialylation. Reduction of the anomeric azido group afforded the sialyl LewisX amine building block. Two molecules of this tetrasaccharide ligand were conjugated to a preformed cyclooctapeptide containing two equidistant l-asparagine units equipped with carboxy-terminated tetraethyleneglycol side chains to give, after deprotection, the target glycopeptide conjugate. Preliminary biological evaluation of the synthesized bivalent sialyl LewisX cyclopeptide conjugate showed only slightly enhanced inhibition of E-selectin binding in spite of the given flexibility of the two linked saccharide determinants.

833. Metabolically Stabilized Non-Viral Gene Delivery Systems

Peptide-based gene delivery systems are composed of synthetic (20 amino acid) peptides derivatized with a targeting oligosaccharide or a polyethylene glycol chain (PEG) 1,2. Ad-mixtures of the glycopeptide and PEG-peptide conjugates were used to form DNA condensates that were stabilized by glutaraldehyde cross-linking through formation of a di-Schiffs base. The degree of metabolic stability is directly related to the degree of glutaraldehyde crosslinking, with metabolic half-lives in mouse liver ranging from 2 hours to 3 days 3.

Oligosaccharide—Peptide Ligation of Glycosyl Thiolates with Dehydropeptides: Synthesis of S‐Linked Mucin‐Related Glycopeptide Conjugates.

A chemoselective strategy for oligosaccharide-peptide ligation is described in which alpha-thio analogues of mucin-related glycoconjugates can be readily accessed through site-selective conjugate addition of complex oligosaccharide thiolates to dehydroalanine-containing peptides. The efficiency of the ligation is highlighted by the rapid convergent assembly of thio-isosteres of the four tumor-associated carbohydrate antigens, T(N), T, ST(N), and 2,6-ST, as a pair of diastereoisomers at the newly formed cysteine stereocenter. The process proceeds in high yield and with complete retention of the alpha-anomeric configuration.

Chemical synthesis of a skeleton structure of sperm CD52-A GPI-anchored glycopeptide.

Going native: A highly convergent synthetic strategy was established to prepare a skeleton structure of sperm CD52 antigen (depicted), which represents the first chemical synthesis of a complex, all natively linked glycosylphosphatidylinositol (GPI)–glycopeptide conjugate.

Oligosaccharide-peptide ligation of glycosyl thiolates with dehydropeptides: synthesis of S-linked mucin-related glycopeptide conjugates.

A chemoselective strategy for oligosaccharide-peptide ligation is described in which alpha-thio analogues of mucin-related glycoconjugates can be readily accessed through site-selective conjugate addition of complex oligosaccharide thiolates to dehydroalanine-containing peptides. The efficiency of the ligation is highlighted by the rapid convergent assembly of thio-isosteres of the four tumor-associated carbohydrate antigens, T(N), T, ST(N), and 2,6-ST, as a pair of diastereoisomers at the newly formed cysteine stereocenter. The process proceeds in high yield and with complete retention of the alpha-anomeric configuration.

A strategy for rational design of fully synthetic glycopeptide conjugate vaccines.

The present study describes a strategy to rationally design fully synthetic glycopeptide conjugate vaccines. Glycopeptide immunogens were constructed by coupling synthetic oligosaccharides comprising repeating units of synthetic 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate (sPRP) to synthetic peptides containing potent T-helper cell determinants and B-cell epitopes of the Haemophilus influenzae type b (Hib) outer membrane proteins (OMPs) P1, P2, and P6. Rabbit immunogenicity studies revealed that some of these fully synthetic glycoconjugates were capable of eliciting high titers of both anti-PRP and anti-OMP immunoglobulin G antibodies. In addition, we systematically investigated the factors which could influence their immunogenicity. We observed that the magnitude of the anti-PRP antibody response markedly depended on the relative spatial orientation of sPRP and T-cell epitopes, the anti-PRP antibody response was enhanced when a multiple antigenic peptide was used as a carrier, the anti-PRP antibody response was optimal for three PRP repeating units, and lipidation of peptide-PRP conjugates had a minimal effect on the magnitude of the anti-PRP antibody response. The results of this study clearly demonstrate that coupling a carbohydrate hapten to a peptide can provide T-cell help and convert it into a T-cell-dependent antigen. The antisera raised against these conjugates were also found to be protective against Hib infection in the infant rat model of bacteremia.

Chemical synthesis of Haemophilus influenzae glycopeptide conjugates.

A simple procedure for conjugating synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b, poly-3-beta-D-ribose-(1,1)-D-ribitol-5-phosphate (sPRP) to linear peptides is described. The procedure consists of (i) reacting the amino group of amino-heptyl sPRP with m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) in phosphate buffer, pH 7.5; (ii) selectively coupling the MBS-modified sPRP to the sulfhydryl group of the cysteine residue of peptides containing functional T-helper cell epitope(s). The glycopeptide conjugates were purified by gel filtration chromatography, biochemically characterized, and elicited protective level of anti-PRP antibody responses in rabbits.