Carbohydrate-based Vaccines Research Articles

Recent advances in carbohydrate-based cancer vaccines.

Cancer is a complex multifactorial disease for which many promising therapeutic strategies such as immunotherapy are emerging. Malignant cells frequently express aberrant cell surface carbohydrates, which differentiate them from normal "healthy" cells. This characteristic presents a window for the development of synthetic carbohydrate antigen-based cancer vaccines which can be recognized by the immune system and can bring about T cell-dependent immune responses. Antibodies generated against the carbohydrate antigens partake in the inactivation of carbohydrate-decorated cancer cells, by slowing down tumor cell growth and inducing cancer cell apoptosis. Novel synthetic strategies for carbohydrate antigens have led to several synthetic cancer vaccine candidates. In the present review, we describe the latest progress in carbohydrate-based cancer vaccines and their clinical evaluation in various cancers.

Synthesis and immunogenicity of the Mycobacterium tuberculosis arabinomannan-CRM197 conjugate.

Lipoarabinomannan (LAM) is a major structural surface component of Mycobacterium tuberculosis. This study describes the synthesis of the well-defined lipoarabinomannan (LAM) specific dodecasaccharide-protein conjugate and immunological studies. Arabinomannan (AM) dodecasaccharide has been efficiently synthesized and covalently conjugated to carrier proteins, including cross reactive mutant (CRM197) diphtheria toxoid and bovine serum albumin (BSA) for novel neoglycoconjugates, creating a potent T-dependent conjugate vaccine. Preliminary mice immunization studies on the neoglycoconjugate revealed that it could give rise to a strong IgG antibody titer in mice at 4.0 μg dose with an aluminum phosphate adjuvant. AM-CRM197 shows potential as an excellent candidate for a new carbohydrate-based vaccine that would be capable of eliciting a protective immune response against tuberculosis.

Glycans in drug discovery.

Glycans are key players in many biological processes. They are essential for protein folding and stability and act as recognition elements in cell-cell and cell-matrix interactions. Thus, being at the heart of medically relevant biological processes, glycans have come onto the scene and are considered hot spots for biomedical intervention. The progress in biophysical techniques allowing access to an increasing molecular and structural understanding of these processes has led to the development of effective therapeutics. Indeed, strategies aimed at designing glycomimetics able to block specific lectin-carbohydrate interactions, carbohydrate-based vaccines mimicking self- and non-self-antigens as well as the exploitation of the therapeutic potential of glycosylated antibodies are being pursued. In this mini-review the most prominent contributions concerning recurrent diseases are highlighted, including bacterial and viral infections, cancer or immune-related pathologies, which certainly show the great promise of carbohydrates in drug discovery.

Chemical synthesis of glycans and applications in carbohydrate-based vaccines

Carbohydrates play important roles in many biological processes. However, the lack of resources of structurally-defined carbohydrates and their weak immunogenicity have always restricted the development of chemical glycobiology especially carbohydrate-based vaccines. The progress in chemical synthesis of carbohydrates and glycoconjugates has greatly promoted the development of carbohydrate-based vaccines including antibacterial vaccines, anti-tumor vaccines and anti-HIV vaccines. The use of one-pot glycosylation strategy and solid-phase glycosylation strategy is able to synthesize oligosaccharides and even polysaccharides more effectively than before. Glycoconjugates produced by combining the carbohydrate antigens and proper protein carriers improve the immunogenicity of carbohydrates. This review covers the general methods and strategies of carbohydrate chemical synthesis, and the recent development of carbohydrate-based vaccines, and also summarizes related work of our group, hoping to offer readers better understanding of chemical glycobiology especially carbohydrate-based vaccines.

Synthetic Glycans and Glycomimetics: A Promising Alternative to Natural Polysaccharides.

A large quantity of polysaccharide-derived conjugate vaccines have been developed to combat various pathogenic infections. Another prominent polysaccharide, heparin, is listed as an essential drug by the World Health Organization (WHO) to treat thrombus. One of their common problems is that they all derive from natural polysaccharides. Specifically, capsular polysaccharides are mainly obtained from bacterial fermentation and unfractionated heparin is extracted from animal tissues such as porcine mucosa. The quality of natural polysaccharides is inconsistent and traces of contamination would cause a disaster. By contrast, the use of chemical or chemoenzymatic methods could provide structurally homogeneous and quality-controlled glycans. To date, large numbers of polysaccharide fragments and their analogues have been synthesized and evaluated. Some of them even showed comparable activities to their corresponding natural polysaccharides. Here, the latest advances in these synthetic glycan analogues ranging from carbohydrate-based vaccines, heparin-related therapeutics and glycomimetics of polysaccharides are summarized.

Interplay of Carbohydrate and Carrier in Antibacterial Glycoconjugate Vaccines.

Bacterial infections are a serious health concern and are responsible for millions of illnesses and deaths each year in communities around the world. Vaccination is an important public health measure for reducing and eliminating this burden, and regions with comprehensive vaccination programs have achieved significant reductions in infection and mortality. This is often accomplished by immunization with bacteria-derived carbohydrates, typically in conjunction with other biomolecules, which induce immunological memory and durable protection against bacterial human pathogens. For many species, however, vaccines are currently unavailable or have suboptimal efficacy characterized by short-lived memory and incomplete protection, especially among at-risk populations. To address this challenge, new tools and techniques have emerged for engineering carbohydrates and conjugating them to carrier molecules in a tractable and scalable manner. Collectively, these approaches are yielding carbohydrate-based vaccine designs with increased immunogenicity and protective efficacy, thereby opening up new opportunities for this important class of antigens. In this chapter we detail the current understanding of how carbohydrates interact with the immune system to provide immunity; how glycoengineering, especially in the context of glycoconjugate vaccines, can be used to modify and enhance immune responses; and current trends and strategies being pursued for the rational design of next-generation glycosylated antibacterial vaccines. Graphical Abstract.

Synthesis of Sialic Acids, Their Derivatives, and Analogs by Using a Whole-Cell Catalyst.

Sialic acids (Sias) are important constituents of cell surface glycans. Ready access to Sias in large quantities would facilitate the development of carbohydrate-based vaccines and small-molecule drugs. We now present a facile method for synthesizing various natural forms and non-natural derivatives or analogs of Sias by using a whole-cell catalyst, which is constructed by adding a plasmid containing necessary enzyme genes into a metabolically engineered strain of Escherichia coli. The flexible substrate tolerance of incorporated enzymes (N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid aldolase) allows the cellular catalyst to convert a wide range of simple and inexpensive sugars into various Sia-related compounds through an easily scalable fermentation process. Further, syntheses using this whole-cell biotransformation in combination with three conventional enzymatic reactions provide a series of complex Sia-containing glycans (sialyloligosaccharides) and their derivatives bearing different substituents. The processes described herein should permit the large-scale and economical production of both Sias and sialyloligosaccharides, and may complement existing chemical and enzymatic strategies.

Recent progress of fully synthetic carbohydrate-based vaccine using TLR agonist as build-in adjuvant

Fully synthetic vaccine, in which one or multi-molecular antigens are conjugated to a synthetic carrier with well-defined chemical structure, is a new direction to develop carbohydrate-based vaccine against cancer and pathogens. Toll like receptor (TLR) agonists with the ability to stimulate immune response have been widely investigated and been applied as build-in adjuvants to construct fully synthetic vaccines. In particular, remarkable progress has been achieved in recent years in the development of vaccines constructed with the agonists of TLR1/2, TLR2/6 and TLR4 and tumor-associated carbohydrate antigens (TACAs). These di-, tri- or multi-component vaccine candidates showed attractive immunological properties. This review highlights recent advances in developing full synthetic carbohydrate antigen based vaccines, with an emphasis on the structure-activity relationships that provide a primary basis for future vaccine design and immunotherapy developing.

Advancing Homogeneous Antimicrobial Glycoconjugate Vaccines.

Since 2004, when the first synthetic glycoconjugate vaccine against the pneumonia and meningitis causing bacterium Haemophilus influenza type b (Hib) approved for human use in Cuba was reported, 34 million doses of the synthetic vaccine have been already distributed in several countries under the commercial name of Quimi-Hib. However, despite the success of this product, no other synthetic glycoconjugate vaccine has been licensed in the following 13 years. As well as avoiding the need to handle pathogens, synthetic glycoconjugates offer clear advantages in terms of product characterization and the possibility to understand the parameters influencing immunogenicity. Nevertheless, large scale application of synthetic sugars has been perceived as challenging because of manufacturing costs and process complexity compared to natural polysaccharides. Chemoenzymatic approaches, one-pot protocols, and automated solid-phase synthesis are rendering carbohydrate production considerably more attractive for industrialization. Here we identify three areas where chemical approaches can advance this progress: (i) chemical or enzymatic methods enabling the delivery of the minimal polysaccharide portion responsible for an effective immune response; (ii) site-selective chemical or enzymatic conjugation strategies for the exploration of the conjugation point in immune responses against carbohydrate-based vaccines, and the consistent preparation of more homogeneous products; (iii) multicomponent constructs targeting receptors responsible for immune response modulation in order to control its quality and magnitude. We discuss how synthesis of bacterial oligosaccharides is useful toward understanding the polysaccharide portion responsible for immunogenicity, and for developing robust and consistent alternatives to natural heterogeneous polysaccharides. The synthesis of sugar analogues can lead to the identification of hydrolytically more stable versions of oligosaccharide antigens. The study of bacterial polysaccharide biosynthesis aids the development of in vitro hazard-free oligosaccharide production. Novel site-selective conjugation methods contribute toward deciphering the role of conjugation sites in the immunogenicity of glycoconjugates and prove to be particularly useful when glycans are conjugated to protein serving as carrier and antigen. The orthogonal incorporation of two different carbohydrate haptens enables the reduction of vaccine components. Finally, coordinated conjugation of glycans and small molecule immunopotentiators supports simplification of vaccine formulation and localization of adjuvant. Synergistic advancement of these areas, combined with competitive manufacturing processes, will contribute to a better understanding of the features guiding the immunological activity of glycoconjugates and, ultimately, to the design of improved, safer vaccines.

Automated glycan assembly using the Glyconeer 2.1 synthesizer

Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure-activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.

NMR line-fitting quantification of polysaccharide N-acylurea-based modification in glycoconjugates of Salmonella Typhi Vi polysaccharide.

The polysaccharides modification via carbodiimide reaction is one of the most applied methods for obtaining conjugated vaccines against Salmonella enterica. However, N-acylurea carbodiimide adduct generated in the process is a critical impurity in carbohydrate-based vaccines. A quantitative NMR method was developed for assessing the N-acylurea carbodiimide adduct impurity. The procedure was based on line-fitting facilities for processing the NMR signals on complex spectra. The method showed good linearity, accuracy and precision under inter-operator variation (relative standard deviation <5%). Copyright © 2017 John Wiley & Sons, Ltd.

IgG Antibody Response Elicited by a Fully Synthetic Two-Component Carbohydrate-Based Cancer Vaccine Candidate with α-Galactosylceramide as Built-in Adjuvant.

A fully synthetic self-adjuvanting cancer vaccine candidate was constructed through covalent conjugation of invariant natural killer T (iNKT) cell ligand α-galactosylceramide (αGalCer) with sialyl Tn (STn), a representative tumor-associated carbohydrate antigen (TACA). This two-component vaccine STn-αGalCer is devoid of antigenic peptide, featuring the well-defined structure with high simplicity. STn-αGalCer showed remarkable efficacy in inducing antibody class switching from IgM to STn-specific IgG. Subtypes of IgG antibody were primarily IgG1 and IgG3.

Cyclopeptide scaffolds in carbohydrate-based synthetic vaccines.

Cyclopeptides have been recently used successfully as carriers for the multivalent presentation of carbohydrate and peptide antigens in immunotherapy. Beside their synthetic versatility, these scaffolds are indeed interesting due to their stability against enzyme degradation and low immunogenicity. This mini-review highlights the recent advances in the utilization of cyclopeptides to prepare fully synthetic vaccines prototypes against cancers and pathogens.

Identification and Design of Synthetic B Cell Epitopes for Carbohydrate-Based Vaccines.

Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward. We here describe how synthetic oligosaccharide epitopes for candidate vaccines can be rationally identified prior to preclinical immunogenicity studies. Epitopes are selected on the basis of their recognition by antibodies associated with protection from disease in humans or small animals. In addition, we show how murine antibody responses to a large oligosaccharide can inform the identification of a minimal B cell epitope that may help designing easy to synthesize vaccine candidates. The procedures, exemplified with a surface carbohydrate of Clostridium difficile, may serve as a guideline for selecting protective oligosaccharide epitopes for vaccines against infectious and malignant diseases.

Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model.

Nanotechnology-based fully synthetic carbohydrate vaccines are promising alternatives to classic polysaccharide/protein conjugate vaccines. We have prepared gold glyco-nanoparticles (GNP) bearing two synthetic carbohydrate antigens related to serotypes 19F and 14 of Streptococcus pneumoniae and evaluated their immunogenicity in vivo. A tetrasaccharide fragment of serotype 14 (Tetra-14), a trisaccharide fragment of serotype 19F (Tri-19F), a T-helper peptide and d -glucose were loaded onto GNP in different ratios. Mice immunization showed that the concomitant presence of Tri-19F and Tetra-14 on the same nanoparticle critically enhanced the titers of specific IgG antibodies toward type 14 polysaccharide compared with GNP exclusively displaying Tetra-14, while no IgG antibodies against type 19F polysaccharide were elicited. This work is a step forward toward synthetic nanosystems combining carbohydrate antigens and immunogenic peptides as potential carbohydrate-based vaccines.

The adjuvant effect of TLR7 agonist conjugated to a meningococcal serogroup C glycoconjugate vaccine

Conjugation of a small molecule immunopotentiator to antigens has been proposed to deliver the ligand to the receptor, localize its action and minimize systemic inflammation. However, the effect of conjugation of Toll like receptor 7 agonists (TLR7a) on the immunogenicity of carbohydrate-based vaccines is unknown.In this study we synthesized an anti-Neisseria meningitidis serogroup C (MenC) glycoconjugate vaccine composed of MenC oligosaccharide antigens covalently linked to the carrier protein CRM197, to which a TLR7a was in turn conjugated. This vaccine was able to activate in vitro the TLR7 comparably to the unconjugated ligand. The magnitude and the quality of the immune response against MenC capsular polysaccharide were evaluated in mice, comparing the MenC-CRM-TLR7a construct to a MenC-CRM197 vaccine, prepared through the same conjugation chemistry and co-administered with the unconjugated TLR7a. A commercially licensed anti-MenC glycoconjugate was used as further control to determine the influence of the coupling approach and the level of carbohydrate incorporation on the anti-MenC immune response. The possible additive effect of co-administration with Alum hydroxide (AlumOH) was also examined.The bactericidal titers against N. meningitidis were in agreement with the elicited anti-carbohydrate IgGs, and unequivocally showed that TLR7a conjugation to CRM197 enhanced the anti-MenC immune response. TLR7a conjugation induced a shift to a Th1 type response, as assessed by the increased IgG2a subclass production, both in the absence and in the presence of AlumOH. The increased immune response was clearly present only in the absence of AlumOH and was less pronounced than the co-administration of a licensed glycoconjugate with a standard dose of TLR7a-phosphonate adsorbed on the inorganic salt. The amount of MenC saccharide that was covalently linked to CRM197 after previous CRM197-TLR7a conjugation resulted in lower responses than achieved with conventional MenC-CRM197 glycoconjugation in the absence of TLR7a. As result, the benefit of the adjuvant conjugation in terms of anti-MenC immune response was jeopardized by the lower saccharide/protein ratio obtained in the MenC-CRM-TLR7a conjugate.While adsorption on AlumOH offers more flexibility in the administered dose of TLR7a, conjugation of the small molecule immunopotentiator could be particularly suited for vaccination routes such as skin delivery, where insoluble aluminum salts cannot be used because of their reactogenicity in this site.

Carbohydrates as T-cell antigens with implications in health and disease.

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease.

Entirely Carbohydrate-Based Vaccines: An Emerging Field for Specific and Selective Immune Responses.

Carbohydrates are regarded as promising targets for vaccine development against infectious disease because cell surface glycans on many infectious agents are attributed to playing an important role in pathogenesis. In addition, oncogenic transformation of normal cells, in many cases, is associated with aberrant glycosylation of the cell surface glycan generating tumor associated carbohydrate antigens (TACAs). Technological advances in glycobiology have added a new dimension to immunotherapy when considering carbohydrates as key targets in developing safe and effective vaccines to combat cancer, bacterial infections, viral infections, etc. Many consider effective vaccines induce T-cell dependent immunity with satisfactory levels of immunological memory that preclude recurrence. Unfortunately, carbohydrates alone are poorly immunogenic as they do not bind strongly to the MHCII complex and thus fail to elicit T-cell immunity. To increase immunogenicity, carbohydrates have been conjugated to carrier proteins, which sometimes can impede carbohydrate specific immunity as peptide-based immune responses can negate antibodies directed at the targeted carbohydrate antigens. To overcome many challenges in using carbohydrate-based vaccine design and development approaches targeting cancer and other diseases, zwitterionic polysaccharides (ZPSs), isolated from the capsule of commensal anaerobic bacteria, will be discussed as promising carriers of carbohydrate antigens to achieve desired immunological responses.

Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.