molecules Review
Recent Advances in the Synthesis of Glycoconjugates for Vaccine Development Cinzia Colombo, Olimpia Pitirollo and Luigi Lay *
ID
Dipartimento di Chimica, Universita’ degli Studi di Milano, via Golgi 19, 20133 Milano, Italy;
[email protected] (C.C.);
[email protected] (O.P.) * Correspondence:
[email protected]; Tel.: +39-025-031-4062
Received: 8 June 2018; Accepted: 11 July 2018; Published: 13 July 2018
Abstract: During the last decade there has been a growing interest in glycoimmunology, a relatively new research field dealing with the specific interactions of carbohydrates with the immune system. Pathogens’ cell surfaces are covered by a thick layer of oligo- and polysaccharides that are crucial virulence factors, as they mediate receptors binding on host cells for initial adhesion and organism invasion. Since in most cases these saccharide structures are uniquely exposed on the pathogen surface, they represent attractive targets for vaccine design. Polysaccharides isolated from cell walls of microorganisms and chemically conjugated to immunogenic proteins have been used as antigens for vaccine development for a range of infectious diseases. However, several challenges are associated with carbohydrate antigens purified from natural sources, such as their difficult characterization and heterogeneous composition. Consequently, glycoconjugates with chemically well-defined structures, that are able to confer highly reproducible biological properties and a better safety profile, are at the forefront of vaccine development. Following on from our previous review on the subject, in the present account we specifically focus on the most recent advances in the synthesis and preliminary immunological evaluation of next generation glycoconjugate vaccines designed to target bacterial and fungal infections that have been reported in the literature since 2011. Keywords: bacterial infections; fungal infections; carbohydrates; glycoconjugates; vaccines
1. Introduction Notwithstanding the great advances of modern medicine, infectious diseases still have a strong impact on public health, both in industrialized and developing countries, due to their significant health-related costs for clinical treatment. In particular, the list of the drug-resistant bacteria is increasing continuously, and novel and more efficient means to prevent microbial infections caused by antibiotic-resistant microorganisms are urgently needed. According to the World Health Organization (WHO) [1], vaccination is the most cost-effective strategy for controlling infections caused by pathogenic microorganisms. Actually, vaccines are able to confer long-term protective immunity on the population and have made possible a great revolution in the 20th century, saving millions of lives. The surface of bacterial pathogens is covered with a dense array of complex glycans, such as lipopolysaccharide of Gram-negative bacteria and the polysaccharide coat (capsular polysaccharides, CPS) of encapsulated bacteria that are crucial protective antigens and major virulence factors. For example, each strain of Streptococcus pneumoniae (the pneumococcus) produces one out of 90 different capsular polysaccharides, which are believed to have been selected as a mechanism to evade the human immune response [2]. All these glycoforms are capable of interacting with the immune system inducing the production of carbohydrate-specific antibodies. They therefore represent attractive targets for vaccine design. Molecules 2018, 23, 1712; doi:10.3390/molecules23071712
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A major drawback of polysaccharide-based vaccines, however, is their limited clinical efficacy. They induce T cell-independent immune responses, featured by poor immunogenicity in children under 5 years of2018, age, elderly and immunocompromised individuals, and fail to generate2conventional Molecules 23,in x FOR PEER REVIEW of 52 B cell-mediated immunological memory. Polysaccharide immunogenicity can be strongly enhanced by drawback of polysaccharide-based vaccines, however, is their limited clinical efficacy. conjugation A tomajor an immunogenic carrier protein, providing T cell-dependent glycoconjugate antigens They induce T cell-independent immune responses, featured by poor immunogenicity in children able to stimulate B cell maturation to memory cells and induce immunoglobulin class switching from under 5 years of age, in elderly and immunocompromised individuals, and fail to generate IgM to polysaccharide-specific IgG. The introduction glycoconjugate vaccines represented one conventional B cell-mediated immunological memory.ofPolysaccharide immunogenicity can be strongly enhanced by conjugation to an immunogenic carrier protein, providing T cell-dependent of the keys for success of vaccination, especially for infants and young children who are the most ablediseases to stimulate cell maturation to memory cellsneeded and induce affected glycoconjugate population byantigens infectious [3–6].B Carbohydrate-based antigens for inclusion immunoglobulin class switching from IgM to polysaccharide-specific IgG. The introduction of in a glycoconjugate vaccine, however, are not readily available from natural sources. In particular, glycoconjugate vaccines represented one of the keys for success of vaccination, especially for infants the isolation and purification of the naturally occurring glycans is still adiseases great challenge that may lead to and young children who are most affected population by infectious [3–6]. Carbohydrateheterogeneous compositions and batch-to-batch variability. A relevant is theavailable toxic endotoxin based antigens needed for inclusion in a glycoconjugate vaccine, however,example are not readily natural sources. In the isolation and (LPS) purification of naturally occurring is lipid A, afrom major component ofparticular, the lipopolysaccharide of Shigella flexneri 2a. Theglycans development of still a great challenge that may lead to heterogeneous compositions and batch-to-batch variability. A LPS-based conjugate vaccines against Shigella flexneri requires careful LPS-detoxification, a technically relevant example is the toxic endotoxin lipid A, a major component of the lipopolysaccharide (LPS) demanding and expensive process which also increases the manufacture costs [7,8]. Hence, the of Shigella flexneri 2a. The development of LPS-based conjugate vaccines against Shigella flexneri development of careful cost-effective, glycoconjugate vaccines basedand on fully synthetic requires LPS-detoxification, a technically demanding expensive processsaccharide which also antigens is gaining growing as demonstrated by development the outstanding success of glycoconjugate the synthetic vaccine increases theimportance, manufacture costs [7,8]. Hence, the of cost-effective, vaccines on fully synthetic saccharide antigens is gainingcompositions, growing importance, as highly Quimi-Hib [9]. based Synthetic glycans, indeed, possess well-defined affording demonstrated by the outstanding success of the synthetic vaccine Quimi-Hib [9]. Synthetic glycans, reproducible biological properties and a better safety profile. In addition, synthetic oligosaccharides indeed, possess well-defined compositions, affording highly reproducible biological properties and can help to elucidate the minimal structure of the microbial polysaccharide, referred to as epitope or a better safety profile. In addition, synthetic oligosaccharides can help to elucidate the minimal antigenicstructure determinant [10], thatpolysaccharide, can ensure production ofepitope a sufficient amount of bactericidal antibodies of the microbial referred to as or antigenic determinant [10], that to confercan long term protective immunity of the host. This step istocrucial forterm the protective design of a new ensure production of a sufficient amount of bactericidal antibodies confer long immunity of the host. step is crucialobtained for the design of afrom new chemical generation synthesis of improved safer source. generation of improved andThis safer vaccines either orand bacterial vaccines obtained either from chemical synthesis or bacterial source. Consequently, glycoconjugates Consequently, glycoconjugates based on chemically well-defined oligosaccharide structures are now based on chemically well-defined oligosaccharide structures are now at the forefront of vaccine at the forefront of vaccine development. development.
f
f
Figure 1. Representation of the monosaccharide building blocks contained in the glycans of pathogen
Figure 1.cell Representation of in the monosaccharide building blocks contained in the glycans of pathogen wall PS accounted this manuscript. cell wall PS accounted in this manuscript.
Over the last years, the synthesis of complex glycans has made significant progress. A variety of synthetic approaches such as automated solid phase synthesis, one-pot programmable synthesis,
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enzymatic and improved synthetic methods have introduced new and elegant ways to provide Molecules 2018, 23, x FOR PEER REVIEW 3 of 52 oligosaccharide antigens with well-defined chemical structure for immunological studies. Meanwhile, the last years, the synthesis of complex glycans madecrystallography, significant progress. A variety of silico improved Over methods for structural elucidation, based onhas X-ray NMR, or in synthetic such as automated solidcarbohydrate-protein phase synthesis, one-pot programmable synthesis, studies, as well approaches as advanced techniques to study interactions (glycoarray, surface enzymatic and isothermal improved synthetic have competitive introduced new and assay) eleganthave waysbeen to provide plasmon resonance, titrationmethods calorimetry, ELISA extensively oligosaccharide antigens with well-defined chemical structure for immunological studies. applied to predict the minimal structural requirements needed for the immunological activity of the Meanwhile, improved methods for structural elucidation, based on X-ray crystallography, NMR, or oligosaccharides. Accordingly, a variety of saccharide fragments reproducing or mimicking the surface in silico studies, as well as advanced techniques to study carbohydrate-protein interactions carbohydrates of pathogens haveresonance, been synthesized, carrier proteins or T ELISA cell peptides, (glycoarray, surface plasmon isothermalcoupled titration to calorimetry, competitive assay) and tested have for their to elicit protective antibodies in animal models. In this regard, thethe present beenability extensively applied to predict the minimal structural requirements neededinfor reviewimmunological we focus on the mostofsignificant advances Accordingly, in the synthesis and preliminary activity the oligosaccharides. a variety of saccharide immunological fragments reproducing or mimicking the surface of pathogens have been synthesized, evaluation of synthetic antibacterial and carbohydrates antifungal glycoconjugate vaccine candidates,coupled appeared in to carrier proteins or T cell peptides, and tested for their ability to elicit protective antibodies in animal the literature from 2011 onwards, following our previous account on the subject [11]. For clarity, models. In this regard, in the present review we focus on the most significant advances in the the diagrammatic representations of the monosaccharide residues [12] illustrated in Figure 1 above are synthesis and preliminary immunological evaluation of synthetic antibacterial and antifungal used throughout this review. glycoconjugate vaccine candidates, appeared in the literature from 2011 onwards, following our previous account on the subject [11]. For clarity, the diagrammatic representations of the 2. Shigella monosaccharide residues [12] illustrated in Figure 1 above are used throughout this review.
The Shigella family includes four different groups of Gram-negative bacteria—S. dysenteriae, 2. Shigella S. sonnei, S. flexneri and S. boydii—each of them comprising different serotypes. Shigella is the The Shigella family includes four different groups of bacteria—S. causative agent of endemic and epidemic shigellosis orGram-negative bacillary dysentery, andysenteriae, invasive S. disease sonnei, S. flexneri and S. boydii—each of them comprising different serotypes. Shigella is the causative of the lower intestine, highly diffused in developing countries and particularly in pediatric population. agent of endemic shigellosis or bacillary dysentery, an invasive diseasetype of the lower The development of a and fullyepidemic synthetic glycoconjugate vaccine against S. dysenteriae 1 is currently intestine, highly diffused in developing countries and particularly in pediatric population. The under investigation, using fragments of the O-antigen of the Shigella LPS. Oligomers up to four development of a fully synthetic glycoconjugate vaccine against S. dysenteriae type 1 is currently repeating units of the tetrasaccharide [α-L-Rha-(1→2)-α-D-Gal-(1→3)-α-D-GlcNAc-(1→3)-α-L-Rha] under investigation, using fragments of the O-antigen of the Shigella LPS. Oligomers up to four were first synthesized and covalently linkedD-Gal-(1→3)-αto human serum albumin (HSA) [13,14]. repeating units of by the Pozsgay tetrasaccharide [α-L-Rha-(1→2)-αD-GlcNAc-(1→3)-αL-Rha] Preliminary studies showed the hexadecasaccharide 4) is the most immunogenic fragment were first synthesized bythat Pozsgay and covalently linked (n to = human serum albumin (HSA) [13,14]. studies showedIgG that in themice hexadecasaccharide (n =upstream 4) is the most immunogenic fragment able toPreliminary elicit anti O-SP-specific [14] and that the residue (non-reducing end) of able to elicit anti O-SP-specific IgG the in mice [14] and that theofupstream residue (non-reducing end) of the synthetic fragments is crucial for immunogenicity these conjugates [15]. synthetic fragments crucial forprevalent the immunogenicity of these [15]. S.the flexneri serotype 2a isisthe most pathogenic strainconjugates in human and a major cause of the S. flexneri serotype 2a is the most prevalent pathogenic strain in human and a major cause of the endemic form of shigellosis in developing countries. The O-antigen of S. flexneri 2a surface LPS is an endemic form of shigellosis in developing countries. The O-antigen of S. flexneri 2a surface LPS is an essential virulence factor and consists of a branched pentasaccharide repeating unit (Figure 2). essential virulence factor and consists of a branched pentasaccharide repeating unit (Figure 2).
Figure 2. Repeating unit of the O-antigen of S. flexneri 2a LPS and synthetic fragments reported
Figure 2. Repeating unit of the O-antigen of S. flexneri 2a LPS and synthetic fragments reported in [16–18]. in [16–18]. In 2005, the Mulard group reported the synthesis of the monomer, dimer and trimer of the pentasaccharide repeating unitreported (AB(E)CD, 2) and conjugation dimer to a universal T cellof the In 2005, the Mulard group theFigure synthesis oftheir the monomer, and trimer peptide epitope, the pan HLA DR-binding epitope (PADRE) [16,17]. Subsequently, the synthesis pentasaccharide repeating unit (AB(E)CD, Figure 2) and their conjugation to a universal T cell of peptide
epitope, the pan HLA DR-binding epitope (PADRE) [16,17]. Subsequently, the synthesis of S. flexneri serotype 2a O-Ag synthetic fragments 1a–18a (Figure 2) was reported [19]. The antigenicity of all
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synthetic fragments 1a–18a was evaluated by ELISA assays, in order to identify the immunogenic determinants recognized by five protective mIgGs specific to serotype 2a O-Ag. None of the mono- or disaccharides was recognized, while the sequence ECD (trisaccharide 9a) was the only one recognized by one mIgG out of five. Tetrasaccharide B(E)CD 13a was recognized by three of the protective mIgG out of five. The minimal sequences for recognition of all mIgGs were pentasaccharides AB(E)CD 16a and B(E)CDA 14a. Following these encouraging results, some selected synthetic oligosaccharides were conjugated to tetanus toxoid (TT) protein and used for immunization studies in mice, leading to the identification of a hit glycoconjugate, 19b, containing the trimer of the pentasaccharide AB(E)CD. Glycoconjugate 19b induced an efficient serotype 2a-specific anti-O-Ag Ab response and it was found to be a functional mimic of the native polysaccharide [20]. Recently, Mulard et al. established a reproducible bioconjugation method for the synthesis of the pentadecasaccharide−TT conjugate 19b, which allowed complete control of the optimal loading [18]. Alum, an adjuvant used in licensed glycoconjugate vaccines like Prevnar 13 or Synflorix, was added to S. flexneri serotype 2a vaccine candidate 19b, which upon immunization was shown to generate a higher and sustained anti-LPS IgG response compared to their nonadjuvanted form [18]. Importantly, Mulard et al. showed that anti-LPS IgG elicited by their synthetic TT conjugate 19b recognized SF2a bacteria and not only purified SF2a LPS [18]. In addition to these promising findings, the influence of O-acetylation of S. flexneri 2a O-Ag fragments on antigenicity was studied by Mulard group [21]. Polysaccharide O-acetylation has been shown to play a key role for many pathogens in inducing functional Ab responses [22–24]. In particular, three diversely O-acetylated S. flexneri 2a O-Ag decasaccharides were synthesized in homogeneous form and their binding to five different protective mAbs was studied, showing some differences in the recognition patterns. Although these data couldn’t provide an exhaustive proof of the role of O-acetylation for S. flexneri 2a O-Ag and of the effect of multiple acetates on the antigen, this work showed that studies using synthetic oligosaccharides may contribute to a better understanding of the antigen-antibody molecular recognition event. 3. Clostridium difficile Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium causing Clostridium difficile infection (CDI), a serious diarrhoeal disease and one of the major cause of hospital-acquired infections (also known as nosocomial infections) in Western countries [25]. The epidemiology of CDI has changed dramatically during this millennium, especially in relation to its clinical presentation, response to treatment and antibiotic resistance [25,26]. In general, after antibiotic treatment that leads to the disruption of the gut microbiota, the intestinal epithelium could be colonized by antibiotic-resistant C. difficile spores, which secrete two toxins (toxin A and toxin B) responsible for the clinical symptoms of CDI. Immune-based strategies based on passive administration of monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent CDI in animal models and in clinical trials have been recently reviewed [27,28]. Concurrently, bacterial surface glycans, such as PS-I and PS-II, have been recently proposed as potential target for vaccine development with the aim of preventing bacterial adhesion and colonization. 3.1. PS-I-Clostridium difficile Recently, Martin et al. reported the synthesis of the pentasaccharide repeating unit of PS-I cell wall polysaccharide of C. difficile ribotype 027 (Figure 3), of its related substructures (compounds 20a–25a, Figure 3) and their immunological evaluation for the identification of the minimal epitope [29]. The synthetic fragments were synthesized from monosaccharide building blocks 26–30, linearly proceeding from the downstream end to the upstream end. In particular, the use of the non participating benzyl group at C-2 of thioglycoside 29 provided the condition for 1,2-cis stereoselective glycosylation of glucoside 30, bearing the linker at the anomeric position. Thioglycoside 29 was functionalized with the orthogonal protecting groups para-bromobenzyl (PBB) ether at C-3 and levulinoyl (Lev) ester at C-4 for installation of the branching point.
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Figure 3. C. difficile PS-I repeating unit, synthetic fragments 20–25 and retrosynthetic analysis reported
Figure 3. C. difficile PS-I repeating unit, synthetic fragments 20–25 and retrosynthetic analysis reported in [29]. in [29].
Glycans 20a–25a, immobilized on microarrays, were screened for antibody recognition with samples from C. difficile patients (stools for IgA and serum IgG). IgAfor and IgG antibodies specific with Glycans 20a–25a, immobilized on microarrays, werefor screened antibody recognition to allfrom glycanC.antigens present(stools in mostfor fecal samples sera,for respectively, of both and samples difficilewere patients IgA and and serum IgG). IgA and patients IgG antibodies control groups. Reconvalescent patients showed highly variable antibody levels and statistically specific to all glycan antigens were present in most fecal samples and sera, respectively, of both higher IgG levels. Pentasaccharide 25a was conjugated to CRM197 (non-toxic mutant of diphtheria patients and control groups. Reconvalescent patients showed highly variable antibody levels toxin) and the resulting glycoconjugate 25b was injected in mice, inducing Ig class switching, affinity and statistically higher IgG levels. Pentasaccharide 25a was conjugated to CRM197 control (non-toxic maturation and producing self-specific antibodies, without eliciting antibodies against two mutant of diphtheria toxin) and the resulting glycoconjugate 25b was injected in mice, inducing oligosaccharides (C. difficile PS-II hexasaccharide and Leishmania lipophosphoglycan capping Ig class switching, affinity maturation and producing self-specific antibodies, without eliciting tetrasaccharide). Interestingly, antibodies raised by glycoconjugate 25b also recognized trisaccharide antibodies two α-Rha-(1→3)-Glc control oligosaccharides PS-II hexasaccharide Leishmania 22a andagainst disaccharide 21a, which (C. was difficile identified as the minimal epitope.and Indeed, αRha-(1→3)-Glc disaccharide-CRM 197 conjugate 21b was able antibodies to induce antibodies recognizing the C. 25b lipophosphoglycan capping tetrasaccharide). Interestingly, raised by glycoconjugate difficile PS-I pentasaccharide 25a. In adisaccharide following work [30], a multivalent presentation of disaccharide also recognized trisaccharide 22a and α-Rha-(1 →3)-Glc 21a, which was identified as the 21a on an oligo(amidoamine) synthetic scaffold [31] was shown to be highly antigenic. In particular, minimal epitope. Indeed, α-Rha-(1→3)-Glc disaccharide-CRM197 conjugate 21b was able to induce a pentavalent presentation of the disaccharide 31 (Figure 3), built on the oligo(amidoamine) backbone antibodies recognizing the C. difficile PS-I pentasaccharide 25a. In a following work [30], a multivalent and displaying a T-cell epitope (amino acids 366–383 of the CRM197 protein) showed increased presentation of disaccharide 21a on an oligo(amidoamine) synthetic scaffold [31] was shown to be antigenicity compared with monovalent 21b, eliciting antibodies against pentasaccharide 25a. A highly antigenic. In particular, a pentavalent presentation of the disaccharide 31 (Figure 3), built on detailed investigation of the glycan-antibody binding was conducted with a combination of different the oligo(amidoamine) backbone and displaying a T-cell epitope (amino acids 366–383 of the CRM197 techniques like glycan microarray, surface plasmon resonance, interaction map, saturation transfer protein) showed increasedand antigenicity with monovalent 21b, eliciting antibodies against difference (STD)-NMR isothermal compared titration calorimetry (ITC). It was demonstrated that the mAbs pentasaccharide 25a. A detailed investigation thethe glycan-antibody was conducted with a mainly interacted with the terminal rhamnoseofand adjacent glucosebinding of the disaccharide 21a and that in pentasaccharide 25a the linkage connecting the two disaccharides is not directly engaged in combination of different techniques like glycan microarray, surface plasmon resonance, interaction binding, although the affinity (KD) increases from micromolar disaccharide 21a to map, antibody saturation transfer difference (STD)-NMR and isothermal titrationfor calorimetry (ITC). It was nanomolar for pentasaccharide 25a. Both glycoconjugates 21b and 25b are currently in preclinical demonstrated that the mAbs mainly interacted with the terminal rhamnose and the adjacent glucose evaluation as novel vaccine candidates against C. difficile.
of the disaccharide 21a and that in pentasaccharide 25a the linkage connecting the two disaccharides is not3.2. directly engaged in antibody binding, although the affinity (KD ) increases from micromolar PS-II-Clostridium difficile for disaccharide 21a to nanomolar for pentasaccharide 25a. Both glycoconjugates 21b and 25b are The chemical synthesis of the hexasaccharide repeating unit (Figure 4) of PS-II cell wall currently in preclinical evaluation as novel vaccine candidates against C. difficile.
polysaccharide of C. difficile ribotype 027, one of the most virulent strains, with two similar synthetic strategies, was reported 3.2. PS-II-Clostridium difficilesimultaneously by two groups in 2011 [32,33].
The chemical synthesis of the hexasaccharide repeating unit (Figure 4) of PS-II cell wall polysaccharide of C. difficile ribotype 027, one of the most virulent strains, with two similar synthetic strategies, was reported simultaneously by two groups in 2011 [32,33].
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Figure 4. C. difficile PS-II repeating unit and retrosynthetic analysis of fragment 32 reported in [32].
Figure 4. C. difficile PS-II repeating unit and retrosynthetic analysis of fragment 32 reported in [32]. The synthesis of PS-II oligosaccharide lacking the phosphate group (compound 32a, Figure 4) carried out by Oberli et al. [32] a retrosynthetic [4+2]the glycosylation tetrasaccharide AB(D)C 33 with 4) was Thewas synthesis PS-II oligosaccharide lacking phosphate (compound 32a, Figure Figure 4. C.of difficile PS-II repeating unitvia and analysisof ofgroup fragment 32 reported in [32]. disaccharide B’C’ 34, starting from monosaccharide building blocks 35–38. Hexasaccharide carried out by Oberli et al. [32] via a [4+2] glycosylation of tetrasaccharide AB(D)C 33 with disaccharide conjugated to CRM (32b)oligosaccharide was used for mice immunization and resulted in the production of IgG4) The synthesis of197PS-II lacking the 35–38. phosphate group (compound 32a, Figure B’C’ 34, starting from monosaccharide building blocks Hexasaccharide conjugated to CRM197 antibodies that bound specifically hapten 32a. In addition, IgA antibodies from the stools of patients was used carriedfor out by Oberli et al. [32] and via aresulted [4+2] glycosylation of tetrasaccharide AB(D)C 33that with (32b) was mice immunization in the production of IgG antibodies diagnosed with supernatant and not the serum, wereblocks chosen because the contact site bound disaccharide B’C’CDI 34,(the starting fromstools, monosaccharide building 35–38. Hexasaccharide specifically 32a. isIn the addition, IgAmucosa) antibodies the stools of patients diagnosed with CDI withhapten C. to difficile intestinal werefrom analyzed. containing conjugated CRM197 (32b) was used for mice immunization and Glycan resulted microarrays in the production of IgG (the supernatant stools, and 32a notwere the serum, becauseAnti-PS-II the contact with C. difficile is hexasaccharide antigen used to were screen chosen patient samples. IgA site antibodies were antibodies that bound specifically hapten 32a. In addition, IgA antibodies from the stools of patients found in the stools of patients diagnosed with CDI, suggesting that the synthetic hexasaccharide the intestinal mucosa) were analyzed. Glycan microarrays containing hexasaccharide antigen diagnosed with CDI (the supernatant stools, and not the serum, were chosen because the contact site 32a could be used in a glycoconjugate vaccine candidate against CDI. were found in the stools of patients were used with to C.screen difficilepatient is thesamples. intestinalAnti-PS-II mucosa) IgA wereantibodies analyzed. Glycan microarrays containing Danieli et al. [33] reported the synthesis of hexasaccharide 39a and its phosphorylated analogue hexasaccharide 32a were to screen hexasaccharide patient samples. Anti-PS-II IgA antibodies were diagnosed with CDI,antigen suggesting thatused the synthetic could be used in a glycoconjugate 40a, starting from disaccharide 41 and tetrasaccharide 42 (or its analogue 43) via a [4+2] convergent found in the stools of CDI. patients diagnosed with CDI, suggesting that the synthetic hexasaccharide vaccine candidate against approach (Figure 5). could beetused in a reported glycoconjugate vaccine candidate against CDI.39a and its phosphorylated analogue Danieli al. [33] the synthesis of hexasaccharide OH Ph Danieli reported the synthesis of hexasaccharide 39a analogue ref 33 and HOet al. [33] RO A its phosphorylated OH Ph OR 40a, starting from disaccharide 41 and tetrasaccharide 43) via aC [4+2] convergent O BnO B' 42 (or its analogue O O O OBn C' HO O O O via B 40a, HO startingO from disaccharide 41 and tetrasaccharide 42 (or its analogue 43) a [4+2] convergent BnO O O HO O AcHN BnO OR BnO OH O HO approach (Figure 5). AcO HO HO SPh O HO O OH OH O OCNHCCl OH 2
approach (Figure 5). HO HO
RO HO HO
O
2
O HO O NHAc
O OR1 OH OH HO OH 1 O a R = (CH ) NH O 2 3 2 O 39 R = HO O HO b R1 = linker-CRM197 40 R = PO AcHN 3H2 OH HO HO O OH OH OH OH O O HO O D HO O O O HO HO NHAc HO OR1 OH OH HO OH O OH OH 1 O HO a R O = (CH2)3NH2 39 HO R=H O O = linker-CRM1971 40 RHO = PO3H2 Ob R1 NHAc A OR C OH B
OH R1 = (CH2)3NH2 51 a D O 1 b R = linker-CRM197 OH HO OH O OH OH O C. 5. HO OFigure O HO O O HO NHAc A OR1 C OH B HO HO
BnO BnO
3 OR1 NHTroc 44 45 46 R1=(CH2)3NHCbz R2 = Bn, OAc Ph ref 33 A 2 Ph C OR OBn D BnO B' O OBn C' D O O B 4 BnO R O OR O O O Ph O O BnO O BnO OO BnO 2 AcO HO SPh SEt BnO O 3 BnO HO O OCNHCCl3 2 R SPh O OR BnO OBn OBn OO OR1 NHTroc OBn BnO 47 3 O 44 45 46 O O RNHTroc O OAc BnO 1 O 2 Ph O ref =(CH2)3NHCbz R 34 BnO 41 C R = Bn, OAc NHTroc A OR1 A B C OAc B OBn O OAc O O Ph O 1 2 3 4 OBn D O SPh O 42 R =(CH2)3NHCbz, O BnO D R =R = Bn, R =OH HO BnO O 1 4O HO O Ph =(CH2)3NHCbz, R2=Ac, R3= CHPh, 43 RR O SEt AcO TrocHN OR1 O BnO BnO49 OMP R4=ClAc BnO 48 3 50 R O OR2 OBn O OBn OBn 47 deprotection R1=(CH2)3NHCbz MP=p-methoxyphenyl O O R3O O BnO O Ph O ref 34 BnO C NHTroc A OR1 A B C OAc B OBn O OAc O O Ph O[33,34]. 1 2fragments 3 4 difficile PSII reported in O SPh O 42 R =(CHsynthetic ) NHCbz, R =R = Bn, R =OH O BnO 2 3 HO BnO HO 43 R1=(CH2)3NHCbz, R2=Ac, R3= CHPh, AcO TrocHN OMP OR1 4 R =ClAc 48 49 50 deprotection R1=(CH2)3NHCbz MP=p-methoxyphenyl
O
O OAc 41
SPh NHTroc
Tetrasaccharide 42 was in turn prepared from monosaccharide building blocks 44−47, by first 51 a R1 = (CH2)3NH2 R1 = linker-CRM 197 assembling the blinear trisaccharide ABC and then inserting the α-Glc D unit [33]. An alternative route Figure 5. C. difficile PSII synthetic fragments reported in [33,34].
Figure 5. C. difficile PSII synthetic fragments reported in [33,34]. Tetrasaccharide 42 was in turn prepared from monosaccharide building blocks 44−47, by first assembling the linear trisaccharide ABC and then inserting the α-Glc D unit [33]. Anblocks alternative route Tetrasaccharide 42 was in turn prepared from monosaccharide building 44–47, by first assembling the linear trisaccharide ABC and then inserting the α-Glc D unit [33]. An alternative route leading to tetrasaccharide 43 starting from building blocks 48, 49 and 50 was also developed [34].
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Of note, the glycosylation with 4,6-O-benzylidene-protected ethylthioglycoside 47 allowed the stereoselective introduction of the 1,2-cis linkage for both routes. Tetrasaccharide AB(D)C 51a, obtained from deprotection of 43, was also synthesized to examine the effect of the branching point Molecules 2018, 23, x FOR PEER REVIEW 7 of 52 of the hexaglycosyl unit in determining the immunogenicity. Sera from mice immunized with the PSII-CRM conjugate were used to check their capability to bind synthetic fragments 39a, 197 to tetrasaccharide 43 starting from building blocks 48, 49 and 50 was also developed [34]. Of40a, 51a. leading Tetrasaccharide showed no binding, while both 39a andethylthioglycoside phosphorylated47fragment note, the 51a glycosylation with 4,6-O-benzylidene-protected allowed 40a the bound stereoselective of the 1,2-cis linkage both routes. Tetrasaccharide AB(D)C to 51a,CRM197 anti-PSII antibodies. introduction In a second experiment, the for three synthetic glycans conjugated obtained from deprotection 43, was also synthesized to examine the effect in of the branching point (compounds 39b, 40b and 51b)ofand native PSII-CRM197 were injected mice and evaluated for of the hexaglycosyl unit in determining the immunogenicity. Sera from mice immunized with the their ability to elicit anti PSII antibodies. Sera were analyzed by ELISA for their content of anti PSII PSII-CRM197 conjugate were used to check their capability to bind synthetic fragments 39a, 40a, 51a. IgG, using PSII-HSA for the coating of the plates. Interestingly, only the glycoconjugates obtained Tetrasaccharide 51a showed no binding, while both 39a and phosphorylated fragment 40a bound from theanti-PSII native polysaccharide and the phosphorylated were able induce IgG 197 antibodies. In a second experiment, the threehexasaccharide synthetic glycans40b conjugated to to CRM antibodies that bound PSIIand and low levels anti-PSII IgM injected antibodies. Tetrasaccharide (compounds 39b, 40b 51b) and nativeof PSII-CRM 197 were in mice and evaluated for51b theirand the ability to elicithexasaccharide anti PSII antibodies. were self-specific analyzed by ELISA for their of induce anti PSIIIgG IgG,nor IgM nonphosphorylated 39bSera elicited antibodies butcontent did not using PSII-HSA for the coating of the plates. Interestingly, only the glycoconjugates obtained from anti-PSII titers. A comparison between the studies of Oberli et al. [32] and Adamo et al. [34] reveals that the native polysaccharide and the phosphorylated hexasaccharide 40b were able to induce IgG the phosphate group on the hexasaccharide repeating unit of PS-II plays a subtle immunological role. antibodies that bound PSII and low levels of anti-PSII IgM antibodies. Tetrasaccharide 51b and the Indeed, nonphosphorylated the phosphate group is not required to raise IgG antibodies production against hexasaccharide 39b elicited self-specific antibodies but did not inducehexasaccharide IgG nor hapten 32a, while it titers. is a prerequisite tobetween elicit antibodies phosphorylated and IgM anti-PSII A comparison the studiesrecognizing, of Oberli et al.besides [32] andthe Adamo et al. [34] reveals that the phosphate group the hexasaccharide repeating unit of PS-II plays suggested a subtle the nonphosporylated hapten, also theonnative PS-II polysaccharide. These findings that immunological role. phosphate is not to raise IgG[34] antibodies production the charged phosphate is Indeed, crucial the to mimic thegroup native PSIIrequired polysaccharide and can be used for the against hexasaccharide hapten 32a, while it is a prerequisite to elicit antibodies recognizing, besides design of carbohydrate antigens as vaccine candidates. the phosphorylated and the nonphosporylated hapten, also the native PS-II polysaccharide. These findings suggested that the charged phosphate is crucial to mimic the native PSII polysaccharide [34] 4. Burkholderia pseudomallei and can be used for the design of carbohydrate antigens as vaccine candidates.
Burkholderia pseudomallei is a Gram-negative environmental bacterium which is widespread in the Burkholderia pseudomallei soil and4.surface water in southeast Asia and northern Australia, causing melioidosis, a serious and often Burkholderia is a Gram-negative bacterium is infection widespreadmimicking in fatal disease presentingpseudomallei acute pulmonary infections,environmental fulminant sepsis and which chronic the soil and surface water in southeast Asia and northern Australia, causing melioidosis, a serious tuberculosis [35]. Antibiotic treatment is usually divided into two phases: a first phase to prevent death and often fatal disease presenting acute pulmonary infections, fulminant sepsis and chronic infection from sepsis and a second phase with the aim of preventing recurrence [36]. This protracted treatment mimicking tuberculosis [35]. Antibiotic treatment is usually divided into two phases: a first phase to is not always mortality rate remains high in Australia to 40% Thailand, preventsuccessful death fromand sepsis and a second phase with the(from aim of15% preventing recurrence [36].in This approaching 90%treatment with septicaemia) For and thismortality reason, rate substantial effort has15% been undertaken to protracted is not always [37]. successful remains high (from in Australia 40% in candidates Thailand, approaching 90% protect with septicaemia) [37]. ForB. this reason, substantial effort hasAmong developtovaccine which would humans against pseudomallei infections [38]. been undertaken to develop vaccine candidates which would protect humans against B. pseudomallei the identified virulence factors [39], the capsular polysaccharide of B. pseudomallei, a homopolymer of infections [38]. Among the identified virulence factors [39], the capsular polysaccharide of B. 2-O-acetyl manno-heptopyranose (Figure 6) has been recently considered for the development of an pseudomallei, a homopolymer of 2-O-acetyl manno-heptopyranose (Figure 6) has been recently effectiveconsidered melioidosis vaccine [40]. for the development of an effective melioidosis vaccine [40].
Figure 6. B. pseudomallei CPS repeating unit and retrosynthetic analysis of compound 52 reported in [37].
Figure 6. B. pseudomallei CPS repeating unit and retrosynthetic analysis of compound 52 reported in [37].
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Of note, B. pseudomallei CPS is expressed as a unique serotype in all reported isolates [41], identical Molecules 2018, 23, x FOR PEER REVIEW 8 of 52 to the CPS of the related bacterium Burkholderia mallei [42]. The synthesis of B. pseudomallei and B. malleiOf CPS is challenging dueCPS to the presence of linkages of the isolates CH2 -extension note, B. pseudomallei is expressed as β-mannoside a unique serotype in alland reported [41], at C-6 (Figure 6). In 2016, Scott et al. [37] reported the first synthesis of hexasaccharide 52a starting identical to the CPS of the related bacterium Burkholderia mallei [42]. The synthesis of B. pseudomallei from key 53,due which was in turnofsynthesized disaccharide 54,CH which and B. disaccharide mallei CPS is fragment challenging to the presence β-mannosidefrom linkages and of the 2wasextension assembled from building 55 and 56,[37] convergently in large scale from common at C-6 (Figure 6). Inblocks 2016, Scott et al. reported theprepared first synthesis of hexasaccharide 52a intermediate 57. This compound was obtained in seven from glycal intermediate 58, which starting from key disaccharide fragment 53, which wassteps in turn synthesized from disaccharide 54,was which was assembled from building blocks 55 and 56, convergently prepared in large scale from synthesized from mannose 59 (Figure 6). The β-mannoside linkages were introduced using an indirect common intermediate 57. This compound was obtained in seven steps from glycal intermediate 58, method, based on stereoselective β-glycosylation (ensured by 2-O-acyl participation on the donor) which by was synthesized from mannose (Figure 6). The β-mannoside linkages werewas introduced followed C-2 epimerization. The latter59step, leading to the manno-configuration, performed using an indirect method, based stereoselective β-glycosylation (ensured by 2-O-acyl participation at the disaccharide level and afteroneach iterative coupling, through a two-step oxidation-reduction on the donor) followed by C-2 epimerization. The latter step, leading to the manno-configuration, was with high stereoselectivity. Hexasaccharide 52a was covalently linked to TT and glycoconjugate 52b, performed at the disaccharide level and after each iterative coupling, through a two-step oxidationupon mice immunization, raised low but detectable levels of IgG/IgM, as determined by ELISA test. reduction with high stereoselectivity. Hexasaccharide 52a was covalently linked to TT and Glycoconjugate 52b, however, was shown to stimulate production of antibodies specific for native glycoconjugate 52b, upon mice immunization, raised low but detectable levels of IgG/IgM, as CPS,determined with high functional activity correlated with protective efficacy, as observed by protection in mice by ELISA test. Glycoconjugate 52b, however, was shown to stimulate production of following a lethal dose ofhigh B. pseudomallei [37]. correlated with protective efficacy, as antibodies specific foradministration native CPS, with functional activity observed by protection in mice following a lethal dose administration of B. pseudomallei [37].
5. Brucella
5.Brucella Brucellais one of the world’s major zoonotic pathogens, causing brucellosis, primarily a disease
of animals, suchisas swine, cattle, andpathogens, goats [43]. Humans are infected by close animal Brucella one of thedogs, world’s majorsheep, zoonotic causing brucellosis, primarily a disease contact or consumption of animal products (raw milk, raw milk products, or raw by meat) of animals, such as swine, dogs, cattle, sheep, and goats [43]. Humans are infected closeinfected animal by bacteria of the genus [44,45]. genus Brucella comprises Gram-negative, and intracellular contact or consumption of The animal products (raw milk, raw milk products, facultative or raw meat) infected by bacteriaand of the the current genus classification [44,45]. The genus Brucella species comprises Gram-negative, facultative and pathogens of recognized is based on phenotypic characteristics, intracellular pathogens and the current classification of recognized is basedThe on phenotypic antigenic variation and prevalence of infection in different animal species hosts [46,47]. disease is not characteristics, antigenic variation and the prevalence of infection in different animal hostsonly [46,47]. The for spread by human-human contact and vaccination of animals appears as the means disease is not spread by human-human contact and the vaccination of animals appears as the only disease eradication by vaccination strategies [48]. The O-antigen polysaccharide domain (OPS) means for disease eradication of by two vaccination [48]. The O-antigen polysaccharide domain of Brucella LPS is a copolymer distinctstrategies homopolysaccharide sequences containing the rare (OPS) of Brucella LPS is a copolymer of two distinct homopolysaccharide sequences containing the sugar 4,6-dideoxy-4-formamido-α-D-mannose (-α-D-Rhap4NFo) [6] and simultaneously expresses two rare sugar 4,6-dideoxy-4-formamido-α-D-mannose (-α-D-Rhap4NFo) [6] and simultaneously antigens, the A and M antigens (Figure 7). expresses two antigens, the A and M antigens (Figure 7).
Figure 7. A and M antigenic determinants in Brucella OPS and oligomers synthesized by Bundle et al. [49].
Figure 7. A and M antigenic determinants in Brucella OPS and oligomers synthesized by Bundle et al. [49].
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Three Brucella antigenic phenotypes A+ M− (A-dominant), A− M+ (M-dominant) and A+ M+ have been identified in Brucella strains [50] and antibodies (IgM) against A and M antigens have been used to detect brucellosis [51,52]. The chemical structure of A and M antigens (Figure 7) was definitively elucidated only recently [53]: a longer inner sequence of α(1,2)-linked residues constitutes the A antigen. A shorter sequence, the M antigen, consists of tetrasaccharide repeating units linked as [α(1,2);α(1,3);α(1,2)] and attached to additional copies of this tetrasaccharide or to the A antigen by an α(1,2) linkage [49]. In 2013, the Bundle group [54] reported the synthesis of pentasaccharide 60a (Figure 7) and nonasaccharide 61a, starting from monosaccharides 62, 63 and 64. The synthetic compounds were tested for antigenicity, after conjugation with bovine serum albumin (BSA) [54]. Glycoconjugate 60b was designed to selectively exhibit the M epitope with limited cross reactivity with A-specific antibodies. The nonasaccharide conjugate (compound 61b), containing A and M epitopes, was designed as a possible universal antigen to detect antibodies in animals or humans infected by B. abortus, B. melitensis, and B. suis. An ELISA test was performed with two monoclonal antibodies (YsT9-1 and Bm10) specific for the Brucella A and M antigens, respectively. Interestingly, nonasaccharide antigen 61a bound A- and M-specific antibodies with equivalent avidity, whereas pentasaccharide 60a displays a preference for the M-specific antibody, as expected. However, pentasaccharide 60a, still displaying α(1,2)-linked residues, retained modest to good binding to A-specific mAbs. This initial result paved the way to produce a glycoconjugate vaccine that would not raise antibodies giving false positive results in diagnostic tests for infection. Indeed, the detection of specific anti-M antibodies would indicate infection by Brucella and not by one of the other closely related bacteria that have PS containing 1,2-linked Rha4NFo or Rha4NAc and are known to induce antibodies reactive in the serological test for brucellosis [55]. In a following work, tetrasaccharide 65a, disaccharide 66a and trisaccharides 67a and 68a (Figure 7) were synthesized to assess the largest and smallest M epitopes [56]. International standard B. abortus serum prepared from cattle experimentally infected with an A-dominant strain bound strongly to disaccharide-BSA conjugate 66b and M tetrasaccharide-BSA conjugate 65b [56]. In addition, 65b and 66b also showed strong binding to M-specific mAbs and weak binding with A-specific mAbs. It was also observed that antibodies raised against exclusively α(1,2)-linked Rhap4NFo did not bind well to the 1,3-linked disaccharide [57]. Further improvement of serodiagnosis of brucellosis came when a tether was introduced at the O-4 of the upstream residue (heptasaccharide 69a, Figure 7) [58]. In particular, conjugate 69b (TT) was used for mice immunization and conjugate 68c (with bovine serum albumin, BSA) to monitor antibody responses by ELISA. Mice immunization with glycoconjugate 69b showed that antibodies to the Brucella A antigen could be produced and that these antibodies didn’t react in diagnostic tests based on the M antigen. These findings were confirmed by the results of immunization studies with the OPS of B. abortus strain S99, which contains 98% α-(1,2) and only 2% α(1,3) linkages conjugated to tetanus toxoid. The OPS was subjected to an oxidation reaction using a procedure that concomitantly oxidized all terminal D-Rhap4NFo residue, essentially destroying the M epitope [58]. Immunization studies in mice showed that antibodies against the A epitope dominated. Taken together, all these studies contributed to identify the main elements for a glycoconjugate vaccine candidate for brucellosis and demonstrated that diagnostics based upon the M or A (terminal) epitopes can discriminate infected from vaccinated animals. 6. Haemophilus influenzae Type b (Hib) Haemophilus influenzae is a Gram-negative bacterium predominantly colonizing the human respiratory tract. H. influenzae strains are divided into two subgroups: unencapsulated strains, also named non-typeable (non-reactive with typing antisera) and encapsulated strains (reactive with typing antisera) comprising six serotypes: a, b, c, d, e and f. In particular, serotype b strains (H. influenzae b, Hib) cause severe diseases including meningitis, pneumonia and septicemia, especially in infants and
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children [59]. Hib CPS consists of a polymer of β-D-ribose-D-ribitol-5-phosphate (PRP) disaccharide, characterized by the presence of a phosphodiester linkage between repeating units (Figure 8). Molecules 2018, 23, x FOR PEER REVIEW
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P
P
Figure 8. Synthesis of QuimiHib.
Figure 8. Synthesis of QuimiHib.
The first generation of Hib vaccines, made with purified polyribosyl-ribitol phosphate, induced
Figure 8. Synthesis of QuimiHib. The first generation Hib vaccines, made with purified phosphate, induced relatively low titers ofof serum antibodies, insufficient to protectpolyribosyl-ribitol children from invasive disease [60], ®, ActHib®, HibTiter®) [61]. Vérez and were replaced by Hib PS-conjugate vaccines (PedVaxHIB relativelyThe lowfirst titers of serum antibodies, insufficient to protect children from invasive disease [60], generation of Hib vaccines, made with purified polyribosyl-ribitol phosphate, induced ®vaccine ® ) [61]. ®Vérez Bencomo et al. by developed the first synthetic glycoconjugate in®2004 [9], QuimiHib and were replaced PS-conjugate (PedVaxHIB , ActHib HibTiter relatively low titers Hib of serum antibodies,vaccines insufficient to protect children from ,invasive disease [60], (compound 70, Figure 8) using a one-pot polycondensation strategy starting from synthetic β-D® ®, HibTiter Bencomo et al.replaced developed first synthetic glycoconjugate vaccine in 2004®)[9], and were by Hibthe PS-conjugate vaccines (PedVaxHIB®, ActHib [61].QuimiHib Vérez ribose-(1,1)-D-ribitol-5-H-phosphonate derivative 71 and the phosphodiester-linked compound 72® (compound 8) using one-pot polycondensation strategy synthetic Bencomo70, et Figure al. developed the afirst synthetic glycoconjugate vaccine in starting 2004 [9], from QuimiHib [9]. Final conjugation to TT gave the fully synthetic glycoconjugate vaccine QuimiHib® [62], which (compound 70, Figure 8) using a one-pot derivative polycondensation starting from synthetic β-Dβ-D-ribose-(1,1)D -ribitol-5-H-phosphonate 71 andstrategy the phosphodiester-linked compound contains a mixture of oligosaccharides with six to eight repeating units on average. Recently, Baek et ® ribose-(1,1)D -ribitol-5-H-phosphonate derivative 71 and the phosphodiester-linked compound 72 72 [9]. conjugation gave the fully197synthetic glycoconjugate vaccine QuimiHib al. Final [62] have reported to theTT synthesis of CRM glycoconjugates of PRP oligosaccharides up to [62], ® [62], which [9]. Final conjugation to TT gave the fully synthetic glycoconjugate vaccine QuimiHib which contains a mixture 73–76, of oligosaccharides with six to eight repeating units on average. Recently, decamers (compounds Figure 9). a mixture of oligosaccharides with six to eight repeating units on average. Recently, Baek et Baek contains et al. [62] have reported the synthesis of CRM197 glycoconjugates of PRP oligosaccharides up to al. [62] have reported the synthesis of CRM197 glycoconjugates of PRP oligosaccharides up to decamers (compounds 73–76, Figure 9).
decamers (compounds 73–76, Figure 9). P
P
P
P
Figure 9. Haemophilus influenzae type b PRP oligosaccharides reported in [63].
Oligosaccharide synthesis was performed via H-phosphonate chemistry starting from Figure 9. Haemophilus influenzae type b PRP oligosaccharides reported in [63]. tetrasaccharide building block 77 and using [4+4]oligosaccharides iterative elongation strategy. Figure 9. Haemophilus influenzae type ab PRP reported in [63]. Tetrameric, hexameric, octameric and decameric PRP fragments were obtained using this iterative approach, Oligosaccharide synthesis was performed via H-phosphonate chemistry starting from followed by the introduction of phosphodiester-linked spacer. Tetrasaccharide 77 was synthesized tetrasaccharide building block was 77 and using a [4+4] iterative elongationchemistry strategy. Tetrameric,from Oligosaccharide synthesis performed from disaccharide 78 (Figure 9), in turn obtained via from H-phosphonate the dithioacetal building blockstarting 79. After hexameric, octameric and decameric PRP fragments were obtained using this approach, tetrasaccharide block 77 and using astudies [4+4] iterative strategy. iterative Tetrameric, conjugationbuilding to CRM197 , immunogenicity with theelongation synthetic glycoconjugates 73–76 hexameric, were followed by the introduction of phosphodiester-linked spacer. Tetrasaccharide 77 was synthesized octameric and decameric PRP fragments were obtained using thistowards iterative followed by the performed in a rabbit model. After immunization, sera IgG levels theapproach, PRP oligosaccharides from disaccharide 78 (Figure 9), in turn obtained from the dithioacetal building block 79. After were determined by glycan array analysis. Tetramer conjugate 7377 and octamer conjugate 75 exhibited introduction of phosphodiester-linked spacer. Tetrasaccharide was synthesized from disaccharide conjugation to CRM197, immunogenicity studies with the synthetic glycoconjugates 73–76 were the highest immunogenicity, most likely indicating that four repeating units are sufficient for 197 , 78 (Figure 9), in turn obtained from the dithioacetal building block 79. After conjugation to CRM performed in a rabbit model. After immunization, sera IgG levels towards the PRP oligosaccharides immunogenicity, while the the hexamer conjugate 74 exhibited lower immunogenicity. This was immunogenicity studies with synthetic performed in75aresult rabbit model. were determined by glycan array analysis. glycoconjugates Tetramer conjugate73–76 73 andwere octamer conjugate exhibited ascribed to the folding of the structures and to their different interaction with the immune system Afterthe immunization, sera IgG levels the PRP oligosaccharides byfor glycan highest immunogenicity, mosttowards likely indicating that four repeatingwere unitsdetermined are sufficient receptors. The authors concluded that glycoconjugates of synthetic Hib PRP are immunogenic in a arrayimmunogenicity, analysis. Tetramer conjugate 73 and octamer 75 exhibited the highest immunogenicity, while the hexamer conjugate 74conjugate exhibited lower immunogenicity. This result was rabbit model and, in particular, tetrameric conjugate 73 is a promising candidate for the design of a ascribed to the folding therepeating structures units and toare their differentfor interaction with the immune system mostnew likely indicating that of four sufficient immunogenicity, while the hexamer glycoconjugate Hib vaccine. receptors. The authors concluded that glycoconjugates of synthetic Hib PRP are immunogenic in a conjugate 74 exhibited lower immunogenicity. This result was ascribed to the folding of the structures rabbit model and, in particular, tetrameric conjugate 73 is a promising candidate for the design of a new glycoconjugate Hib vaccine.
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and to their different interaction with the immune system receptors. The authors concluded that glycoconjugates of synthetic Hib PRP are immunogenic in a rabbit model and, in particular, tetrameric conjugate 73 is a promising candidate for the design of a new glycoconjugate Hib vaccine. 7. Streptococcus pneumoniae S. pneumoniae, a Gram-positive organism, is a major cause of pneumonia, otitis media, meningitis and septicemia. Various virulence determinants of pneumococci have been identified including the highly variable capsular polysaccharide (CPS), pneumolysin toxin and surface lectins. Bentley and colleagues have determined the DNA sequence of the capsular biosynthesis genes for all 90 serotypes (ST) of S. pneumoniae and found that each serotype has a different CPS composition [2,64]. The first generation carbohydrate-based vaccine PPV23 (Pneumovax® , Merck) containing the 23 most prevalent serotypes is available in the United States and in Europe, although conflicting data about its efficacy have been reported [65]. To improve the immunogenicity, glycoconjugate vaccines like PCV7 (Prevnar® , containing PS from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), PCV13 (Prevnar 13™, containing PS from serotypes 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 1, 3 and 5) and PCV10 (GlaxoSmithKline’s Synflorix™, containing PS from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) have been licensed and commercialized. Although S. pneumoniae CPS-based glycoconjugate vaccines are in current routine immunization programs and notwithstanding the increased coverage of strains, diseases caused by serotypes not included in the above vaccines can increase in the long run [64,66]. Recent efforts have been dedicated to the synthesis of antigens from S. pneumoniae serotypes not included in licensed formulations. Of note, glycoconjugates from synthetic fragments of S. pneumoniae serotype 8 have been tested in coformulation with PCV13, as reported in Section 7.6. Recently, the synthesis of the hexasaccharide repeating unit of S. pneumoniae serotype 12F, also not included in marketed formulations, has been reported by Seeberger et al. [67]. Meanwhile, alternative and combined approaches are emerging for vaccine development, based, for instance, on immunization with a combination of bacterial lectins and surface polysaccharides. In a recent study, the surface polysaccharide serotype 6B (PS6B) of S. pneumoniae was conjugated to a recombinant pneumococcal surface protein A (lectin rPspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, showing the ability of the novel conjugate to induce production of functional anti-rPspA1 and anti-PS6B antibodies [68]. 7.1. S. pneumoniae Serotype 1 S. pneumoniae serotype 1 (ST1) CPS (Figure 10) contains the rare monosaccharide 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-AAT) bearing a free amine at C-4. Synthetic fragments of ST1 CPS have been reported by Wu et al. [69], Christina et al. [70] and Schumann et al. [71]. In particular, Schumann et al. also contributed to the identification of the protective epitope of ST1 CPS. ST1 is one of the serotypes difficult to target by vaccination due to the low levels of functional antibodies induced by licensed glycoconjugate vaccines. This was recently [72] ascribed to the concealment of the protective epitope during chemical activation and conjugation to carrier protein. Indeed, conjugation strategies by means of reductive amination (PCV13) or 1-cyano-4-dimethylaminopyridine activation chemistry (PCV10) could lead to partial destruction of the D-AAT moieties by reaction with the free amines on this rare monosaccharide. To confirm this assumption Schumann et al. [72] synthesized and tested fragments of ST1 CPS and of the closely related Bacteroides fragilils PS A1 CPS (Figure 10). Synthetic oligosaccharides 80–85 were then subjected to glycan microarray analysis of ST1- and PS A1-directed antisera. Trisaccharide 80 bound to antibodies contained in ST1 typing serum, while disaccharide 82, missing the D-AAT moiety, was bound in a much lower extent, revealing the importance of D-AAT for immune recognition. Neither the PS A1 repeating unit 81 nor D-AAT alone 83 or galacturonic acid alone 84 were bound. Trisaccharide 80 was then conjugated to CRM197 (glycoconjugate 86) by reaction with the thiol group, thus preserving the amino group of D-AAT.
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f
f
Figure 10. Repeating units of Streptococcus pneumoniae 1 CPS and Bacteroides fragilils PS A1 CPS and
Figure 10. synthetic Repeating units of Streptococcus pneumoniae 1 CPS and Bacteroides fragilils PS A1 CPS and glycan fragments reported in [72]. synthetic glycan fragments reported in [72]. Immunization studies in rabbit models showed that glycoconjugate 86 elicited a higher immune response against trisaccharide 80, D-AAT 83 as well as ST1 CPS compared to PCV13 or CRM197 alone. Immunization studies in rabbit models that glycoconjugate 86vitro elicited higher The antibacterial properties of sera againstshowed glycoconjugate 86 were evaluated in and inavivo. In immune response against trisaccharide 80,revealed D -AAT that 83 as well as in ST1 CPS compared to PCV13 or CRM197 alone. particular, flow cytometry antibodies sera from glycoconjugate 86-immunized rabbits bound better toof ST1 bacteria than glycoconjugate sera from PCV13-immunized rabbits. Bacterial binding The antibacterial properties sera against 86 were evaluated in vitro and in vivo. correlated with serum opsonophagocytic killing capacities. Mice were passively immunized with In particular, flow cytometry revealed that antibodies in sera from glycoconjugate 86-immunized serum of rabbits immunized with glycoconjugate 86 and then transnasally infected with ST1 rabbits bound better showing to ST1 fewer bacteria than sera than frommice PCV13-immunized rabbits. Bacterial pneumococci, bacterial colonies pretreated with sera from PCV13 or CRM197 binding correlatedalone-immunized with serum opsonophagocytic killing capacities. Mice were passively86immunized with rabbits. Given the importance of these findings, glycoconjugate is now in preclinical development for inclusion in vaccines covering multiple with ST1 serum of advancing rabbits immunized with glycoconjugate 86semisynthetic and then transnasally infected pneumococcal serotypes [72]. Interestingly, this work further demonstrates that the use of synthetic pneumococci, showing fewer bacterial colonies than mice pretreated with sera from PCV13 or oligosaccharide antigens may be crucial to unveil hidden protective epitopes by means of siteCRM197 alone-immunized rabbits. Given the importance of these findings, glycoconjugate 86 is selective protein conjugation.
now advancing in preclinical development for inclusion in semisynthetic vaccines covering multiple 7.2. S. serotypes pneumoniae Serotype 2 pneumococcal [72]. Interestingly, this work further demonstrates that the use of synthetic ST2 is one ofmay “nonvaccine serotype”, i.e., hidden not covered by licensed PCVs based on capsular oligosaccharide antigens be crucial to unveil protective epitopes by means of site-selective polysaccharides. It is one of the main cause of invasive pneumococcal diseases (IPD) responsible for protein conjugation.
pneumonia, septicemia, meningitis, and otitis media in many countries in Asia [73] and Central America [74]. The structure of ST2 CPS is composed of a hexasaccharide repeating unit illustrated in 7.2. S. pneumoniae Serotype 2 Figure 11 [75]. Emmadi et al. [76] reported the synthesis of thecovered repeatingby unitlicensed of ST2 CPS and of series on of capsular ST2 is one of “nonvaccine serotype”, i.e., not PCVs based synthetic glycans containing portions of the ST2 CPS (compounds 87a–93a), in order to identify the polysaccharides. It is one of the main cause of invasive pneumococcal diseases (IPD) responsible protective oligosaccharide epitope. Hexasaccharide 89a (one repeating unit) was synthesized from for pneumonia, septicemia, and otitis strategy media (Figure in many in Asia [73] and Central disaccharides 94, 95, 96meningitis, via a [2+2+2] glycosylation 11). countries These disaccharide units were in turn L-rhamnose and D-glucose blocks 97–102 (Figure 11). Theillustrated βAmerica [74]. Thesynthesized structurefrom of ST2 CPS is composed ofbuilding a hexasaccharide repeating unit in rhamnosidic linkage in 94 was incorporated by installing a remote C3 picoloyl group on rhamnosyl Figure 11 [75]. thioglycoside 97 for hydrogen-bond-mediated aglycon delivery. The 1,2-cis linkage between glucose Emmadi et al. [76] reported the synthesis of the repeating unit of ST2 CPS and of series of synthetic building blocks 101 and 102 was formed by in situ anomerization, by converting 102 to the glycans containing portions the ST2and CPS (compounds 87a–93a), in presence order toofidentify the protective corresponding glycosylofbromide then by reatcion with 101 in the TBAI. Glycan microarrays containing oligosaccharide fragments 87a–93a were used to synthesized screen human from and rabbit oligosaccharide epitope. Hexasaccharide 89a (one repeating unit) was disaccharides sera specific to serotype 2 CPS and to identify epitope hits. These experiments demonstrated that the 94, 95, 96 via a [2+2+2] glycosylation strategy (Figure 11). These disaccharide units were in turn
synthesized from L-rhamnose and D-glucose building blocks 97–102 (Figure 11). The β-rhamnosidic linkage in 94 was incorporated by installing a remote C3 picoloyl group on rhamnosyl thioglycoside 97 for hydrogen-bond-mediated aglycon delivery. The 1,2-cis linkage between glucose building blocks 101 and 102 was formed by in situ anomerization, by converting 102 to the corresponding glycosyl bromide and then by reatcion with 101 in the presence of TBAI. Glycan microarrays containing oligosaccharide fragments 87a–93a were used to screen human and rabbit sera specific to serotype 2 CPS and to identify epitope hits. These experiments demonstrated that the α-D-GlcA-(1→6)-α-D-Glc-(1→2) branch is important to have strong specific antibody binding. Hexasaccharide 89a was conjugated to CRM197
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α-D-GlcA-(1→6)-α-D-Glc-(1→2) branch is important to have strong specific antibody binding. Hexasaccharide 89a was conjugated to CRM197 and used for mice immunization producing very high and used for mice immunization producing very high titers of CPS-specific opsonizing antibodies titers of CPS-specific opsonizing antibodies that efficiently fix complement and promote killing of that efficiently fix complement and promote killing of pneumococci by phagocytic activity. An in vivo pneumococci by phagocytic activity. An in vivo experiment to evaluate the vaccine involved experiment to evaluate the vaccine involved subcutaneous immunization of mice that were infected subcutaneous immunization of mice that were infected with highly virulent ST2 strain NCTC7466. with highly virulent ST2 strain NCTC7466. Neoglycoconjugate hexasaccharide-CRM197 89b stimulated Neoglycoconjugate hexasaccharide-CRM197 89b stimulated a T cell-dependent B cell response that a T cell-dependent B cell response that induced CPS-specific antibodies resulting in the reduction of induced CPS-specific antibodies resulting in the reduction of the bacterial infection in lung tissues the bacterial infection in lung tissues and blood. and blood.
Figure 11. Repeating unit of Streptococcus pneumoniae 2 CPS and synthetic ST2 glycan fragments Figure 11. Repeating unit of Streptococcus pneumoniae 2 CPS and synthetic ST2 glycan fragments reported in [76]. reported in [76].
7.3. S. Pneumoniae Serotype 3 7.3. S. pneumoniae Serotype 3 The commercial anti-pneumococcal glycoconjugate vaccine PCV13 includes S. pneumoniae The commercial anti-pneumococcal glycoconjugate vaccine PCV13 includes S. pneumoniae serotype 3 (ST3). However, ST3 glycoconjugate contained in PCV13 has shown an atypical serotype 3 (ST3). However, ST3 glycoconjugate contained in PCV13 has shown an atypical immunogenicity, ascribed to the abundant CPS expression on the capsule and to a weakened booster immunogenicity, ascribed to the abundant CPS expression on the capsule and to a weakened booster response leading to hyporesponsiveness (inability of the individual to mount an immune response response leading to hyporesponsiveness (inability of the individual to mount an immune response after booster vaccination) [77,78]. Indeed, the levels of pre-existing ST3-specific antibody were found after booster vaccination) [77,78]. Indeed, the levels of pre-existing ST3-specific antibody were found to be negatively correlated with the B cell memory response to a booster dose of PCV13 containing to be negatively correlated with the B cell memory response to a booster dose of PCV13 containing ST3 glycoconjugate [79]. This behavior has been associated with a lack of protection against acute ST3 glycoconjugate [79]. This behavior has been associated with a lack of protection against acute otitis media [80]. As a consequence, one can assume that pure synthetic antigenic structures, designed otitis media [80]. As a consequence, one can assume that pure synthetic antigenic structures, designed on the basis of antibody binding specificities, could improve immunogenic properties of ST3 CPS on the basis of antibody binding specificities, could improve immunogenic properties of ST3 CPS conjugates. Synthetic oligosaccharides based on ST3 CPS repeating units have been already reported conjugates. Synthetic oligosaccharides based on ST3 CPS repeating units have been already reported by Benaissa-Trouw et al. in 2001 [80] and they have been proven to protect mice against lethal by Benaissa-Trouw et al. in 2001 [80] and they have been proven to protect mice against lethal intraperitoneal challenge with ST3 pneumococci. Recently, Parameswarappa et al. [81] reported the intraperitoneal challenge with ST3 pneumococci. Recently, Parameswarappa et al. [81] reported the synthesis of a library of oligosaccharides, compounds 103a–110a and their corresponding CRM197 synthesis of a library of oligosaccharides, compounds 103a–110a and their corresponding CRM197 conjugates 103b–110b (Figure 12). conjugates 103b–110b (Figure 12).
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Figure 12. Repeating unit of Streptococcus pneumoniae 3 CPS and synthetic ST3 glycan fragments Figure 12. Repeating unit of Streptococcus pneumoniae 3 CPS and synthetic ST3 glycan fragments reported in [81]. reported in [81].
The synthesis of the fragments was achieved from disaccharide 111, in turn obtained from two The synthesis of theglucose fragments was achieved from 111, in turn obtained from two differentially protected building blocks 112 anddisaccharide 113. Tetrasaccharide 110a was synthesized differentially protected glucose building blocks 112 and 113. Tetrasaccharide 110a was synthesized with a [2+2] strategy in 13% overall yield from 112 and 113. The synthetic ST3 oligosaccharides with a [2+2] strategy the in 13% overall yield from 112 and 113. The12). synthetic oligosaccharides potentially contained minimal protective glycan epitope (Figure GlycanST3 arrays containing the potentially contained theused minimal protective glycan epitope (Figure 12). Glycanthe arrays containing different fragments were to screen human sera for antibodies and to define recognition site the different fragments were used to screen human sera for antibodies and to define the recognition of two protective ST3-specific monoclonal antibodies (mAbs). Tetrasaccharide 110a contains the site of two epitope protective ST3-specific monoclonal antibodies (mAbs). Tetrasaccharide 110a contains the protective of both mAbs and was selected for further immunogenicity studies. The CRM 197 protective epitope of both mAbs and was selected for further immunogenicity studies. The CRM 197 conjugate 110b elicited protective immunity as evidenced by opsonophagocytosis assays and mice conjugate 110bexperiments elicited protective as evidenced bycaused opsonophagocytosis assays and immunization againstimmunity experimental pneumonia by transnasal infection withmice ST3 immunization experiments against experimental pneumonia caused by transnasal infection with strain PN36. Formulation of the protective epitope has to be further evaluated to elicit optimal longST3 strain PN36. Formulation of the protective epitope has to be further evaluated to elicit optimal term immunity. long-term immunity. The synthesis of ST3 CPS oligosaccharides 114–117 (Figure 13) has been recently reported by The synthesis of ST3 CPS oligosaccharides has been recently reported by Xiong et al. [82] These oligosaccharides were also 114–117 designed(Figure to have13) different sugar residues, Glc (114 Xiong et al. [82] These oligosaccharides were also designed to have different sugar residues, Glc and 116) or GlcA (115 and 117) at the upstream end. As an example, heptasaccharide 116 was (114 and 116)byora GlcA (115 and 117) atstrategy the upstream end. As an example, heptasaccharide 116 was synthesized 3+[2+2] glycosylation from trisaccharide 118 and disaccharides 119 and 120, synthesized by a 3+[2+2] glycosylation strategy from trisaccharide 118 and disaccharides 119 and 120, all achieved from common precursor 121. Hexasaccharide 115 was synthesized by a [2+2]+2 strategy all achieved from common precursor 115were was designed synthesized a [2+2]+2 from disaccharides 122, 123 and 120.121. TheHexasaccharide oligosaccharides to by expose a freestrategy amino from disaccharides 122, 123 and 120. The oligosaccharides were designed to expose a free amino group group at their downstream ends (114a–117a) to allow conjugation with tetanus toxoid (TT) (114b– at their downstream ends (114a–117a) to allow conjugation with tetanus toxoid (TT) (114b–117b) and 117b) and BSA (114c–117c) carrier proteins. [83]. TT conjugates 114b–117b and free oligosaccharides BSA (114c–117c) carrier proteins. [83]. TT conjugates 114b–117b and free oligosaccharides 114a–117a 114a–117a were injected in mice and the obtained antisera were analyzed by ELISA using BSA were injected in miceasand the obtained antisera derived were analyzed byimmunized ELISA using BSA conjugates 114c–117c capture antigens. Antisera from mice with TT conjugates conjugates 114c–117c as capture antigens. Antisera derived from mice immunized with TT conjugates 114b–117b contained significantly higher specific antibodies compared to 114a–117a. In 114b–117b particular, contained significantly higher specific antibodies compared to 114a–117a. In particular, antibody antibody titers induced by 114b and 115b were significantly higher than those induced by 116btiters and induced by 114bthat andthe 115b werelength significantly than those induced by 116b the andimmunological 117b, showing 117b, showing chain of ST3higher CPS oligosaccharides influences that the chain of ST3oligosaccharides CPS oligosaccharides the immunological properties andlength that longer are notinfluences necessarily better haptens. properties and that longer oligosaccharides are not necessarily better haptens.
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Ph O
AcO AcO OH HO HO
O
HOOC HO O
OH
O O O
OAc
BnO O O OBz ClAcO 118
OH O OH
OR
116
Ph
OH n
114. n = 2 116. n = 3
STol
OBz
+
O
O HO
O
a. R = (CH2)3NH2 b. R = linker-TT c. R = linker BSA
Ph
O O ClAcO
O O ClAcO
BnO O OBzClAcO O
119 + BnO O O OBz ClAcO 120
Ph O
STol
O O HO
OBz
O
STol
OH 121
O
OR1
OBz R1 = (CH2)3N3
Ph HOOC HO O H
OH O OH
O HO
O OH
OR m
115. m = 3 a. R = (CH2)3NH2 117. m = 4 b. R = linker-TT c. R = linker BSA
O O BzO
115 Ph
O O HO
BnO O OBz BzO 122 + BnO O O OBz BzO O
+ Ph
O O ClAcO
O OBz
123 BnO O ClAcO 120
O
STol
OBz
O
STol
OBz
O
OR1
OBz R1 = (CH2)3N3
Figure 13. Synthesis of ST3 oligosaccharides and glycoconjugates from [82].
Figure 13. Synthesis of ST3 oligosaccharides and glycoconjugates from [82].
7.4. S. pneumoniae Serotype 4
7.4. S. pneumoniae Serotype 4
The CPS of S. pneumoniae serotype 4 (ST4) contains a rare and labile substituent, the trans-2,3-(S) cyclic pyruvate ketal modified galactose (residue A, aFigure 12).labile The substituent, ST4 repeating is a The CPS of S. pneumoniae serotype 4 (ST4) contains rare and theunit trans-2,3-(S) tetrasaccharide made of [3)-βD -ManpNAc-(1→3)-αL -FucpNAc-(1→3)-αD -GalpNAc-(1→4)-αDcyclic pyruvate ketal modified galactose (residue A, Figure 12). The ST4 repeating unit is a tetrasaccharide Galp-2, 3-(S)-Pyr-(1→] (Figure 14). made of [3)-β-D-ManpNAc-(1→3)-α-L-FucpNAc-(1→3)-α-D-GalpNAc-(1→4)-α-D-Galp-2, 3-(S)-Pyr-(1→] Recently, the Seeberger group [84,85] reported the synthesis and immunological evaluation of (Figure 14). fragment 124a, corresponding to the repeating unit, and shorter oligomers 125a–131a with and Recently, the Seeberger group [84,85] reported the synthesis and immunological evaluation of without the pyruvate ketal, demonstrating the importance of the trans-2,3(S)-pyruvate ketal in the fragment corresponding the synthetic repeatingfragments unit, and shorter oligomers 125a–131a and without ST4 124a, epitope. In particular,tothe were obtained with a linear with glycosylation the pyruvate importance of the ketal in thelinkages, ST4 epitope. approachketal, from demonstrating building blocks the 132–135 (Figure 13). Of trans-2,3(S)-pyruvate note, for the installation of 1,2-cis In particular, the synthetic fragments were obtained with a linear glycosylation approach from building glycosylation of galactose 135 with donor 134 occurred with good stereoselectivity of the newly glycosidic = 7:1). other hand, of the final β-manno linkage in blocksformed 132–135 (Figurelinkage 13). Of(α:β note, for On thethe installation ofinstallation 1,2-cis linkages, glycosylation of galactose unit D was accomplished using angood indirect two-steps method. exclusive β-glucosylation 135 with donor 134 occurred with stereoselectivity ofIndeed, the newly formed glycosidicwas linkage 132 using NIS and of TfOH promoters. following 2-OH activation and (α:β =achieved 7:1). Onwith the donor other hand, installation the as final β-mannoThe linkage in unit D was accomplished amination established the desired manno configuration at C-2. Glycan arrays showed that ST4using an indirect two-steps method. Indeed, exclusive β-glucosylation was achieved with donor 132 directed antibodies in the human reference serum (serum 007sp) [85] recognized both pyruvateusing NIS and TfOH as promoters. The following 2-OH activation and amination established the dependent and pyruvate-independent epitopes. Oligosaccharide 124a showed the highest antibody desired manno configuration at C-2. Glycan arrays that ST4-directed antibodies in the human affinity and cross-reactivity to ST4 CPS in miceshowed and humans immunized with the natural CPS. reference serum (serum 007sp) [85] recognized both pyruvate-dependent and pyruvate-independent Human serum 007sp contains antibodies recognizing also non-pyruvalated oligosaccharides 129a epitopes. Oligosaccharide 124a showedthat the non-pyruvalated highest antibody affinitycould and be cross-reactivity to ST4 CPS and 131a. Thus, it was hypothesized epitopes present in the natural in mice and humans immunized with thecould natural CPS. Human serum 007sp contains antibodies CPS, although non-pyruvalated epitopes be less immunogenic than pyruvalated epitopes, as indicated by non-pyruvalated lower antibody binding signals to 129a andand 131a compared 124a.hypothesized To verify this that recognizing also oligosaccharides 129a 131a. Thus, to it was behavior, twoepitopes selected CRM 197 be conjugates ST4 oligosaccharides, 129b and 131b, non-pyruvalated could presentofinnon-pyruvalated the natural CPS, although non-pyruvalated epitopes were used for mice immunization. The raised antibodies did not recognize the natural polysaccharide could be less immunogenic than pyruvalated epitopes, as indicated by lower antibody binding signals on the surface of ST4 bacteria. This result confirmed that the pyruvate motif on the oligosaccharide to 129a and 131a compared to 124a. To verify this behavior, two selected CRM197 conjugates of is needed for cross-reactivity with the native CPS.
non-pyruvalated ST4 oligosaccharides, 129b and 131b, were used for mice immunization. The raised antibodies did not recognize the natural polysaccharide on the surface of ST4 bacteria. This result confirmed that the pyruvate motif on the oligosaccharide is needed for cross-reactivity with the native CPS.
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Figure 14. Repeating unit of ST4 CPS and synthesis of ST4 CPS fragments in the pyruvalated form
Figure 14. Repeating unit of ST4 CPS and synthesis of ST4 CPS fragments in the pyruvalated form and and the nonpyruvalated variant, as reported in [84]. the nonpyruvalated variant, as reported in [84]. 14. Repeating unit of ST4 CPS and synthesis of ST4 CPS fragments in the pyruvalated form 7.5. S.Figure pneumoniae Serotype 5
and the nonpyruvalated variant, as reported in [84]. 7.5. S. pneumoniae Serotype 5
Serotype 5 (ST5) is the fifth most prevalent serotype of S. pneumoniae and is included in the PCV10 and PCV13 repeating unit serotype (Figure 15) contains a branched N-acetyl-Lin - the 7.5. S. pneumoniae Serotype 5 CPS Serotype 5 (ST5) is[86]. theST5 fifth most prevalent of[87] S. pneumoniae and is included fucosamine ( L -FucpNAc) linked to D -glucose ( D -Glc) and D -glucuronic acid ( D -GlcA) and two rare PCV10 and PCV13 [86]. isST5 repeating unitserotype (Figureof15) [87] contains Serotype 5 (ST5) the CPS fifth most prevalent S. pneumoniae and ais branched included inN-acetylthe deoxyamino sugars: the ketoamino sugar 2-acetamido-2,6-dideoxyD-xylose-hexos-4-ulose (Sugp) L -fucosamine ( L -FucpNAc) linked to D -glucose ( D -Glc) and D -glucuronic acid (D-GlcA) and PCV10 and PCV13 [86]. ST5 CPS repeating unit (Figure 15) [87] contains a branched N-acetylL- two and the N-acetyl-L-pneumosamine (L-PneupNAc), which is α(1→2) linked to D-GlcpA. During CPS rare deoxyamino the linked ketoamino sugar 2-acetamido-2,6-dideoxyD -xylose-hexos-4-ulose (Sugp) fucosamine (Lsugars: -FucpNAc) to D-glucose (D-Glc) and D-glucuronic acid (D-GlcA) and two rare isolation and purification for the production of the glycoconjugate vaccine, the keto group of Sugp deoxyamino the ketoamino sugar 2-acetamido-2,6-dideoxy-xylose-hexos-4-ulose (Sugp) CPS and the N-acetyl-sugars: L -pneumosamine (L-PneupNAc), which is α(1→2)Dlinked to D-GlcpA. During can be partially or fully reduced to form a mixture of ST5 CPS components with decreased and and the N-acetylL-pneumosamine (L-PneupNAc), which is α(1→2)vaccine, linked to D-GlcpA. During CPS can isolation purification for the production the glycoconjugate the keto group of Sugp immunogenicity compared with the native of ST5 CPS [88]. isolation and purification for the production of the glycoconjugate vaccine, the keto group of Sugp be partially or fully reduced to form a mixture of ST5 CPS components with decreased immunogenicity can be partially or fully reduced to form a mixture of ST5 CPS components with decreased compared with the native ST5 CPS [88]. immunogenicity compared with the native ST5 CPS [88].
Figure 15. Repeating unit of ST5 CPS and synthetic glycan fragments reported in [89].
Recently, Lisboa et al. [89] reported the synthesis of ST5 CPS fragments 136a–138a starting from Figure 15. Repeating unit of ST5 CPS and synthetic glycan fragments reported in [89]. six differentially protected unit monosaccharide building blocks 139–144 (Figure 15). in In[89]. particular, Figure 15. Repeating of ST5 CPS and synthetic glycan fragments reported L-fucosamine acceptor 143 and L-pneumosamine donor 144 were both synthesized from L-fucal 145 Recently, Lisboa et al. [89] reported the synthesis of ST5 CPS fragments 136a–138a starting from via an azido-phenylselenation reaction on the double bond. Among the oligomers synthesized, six differentially protected building blocks 139–144 (Figure 15).136a–138a In particular, Recently, Lisboa et al. [89]monosaccharide reported the synthesis of ST5 CPS fragments starting oligomer 136a contains N-acetylD-quinovosamine (A’, D-QuiNAc) in place of of Sugp (A), displaying L -fucosamine acceptor 143 and L -pneumosamine donor 144 were both synthesized from L-fucal 145 from asix differentially protected monosaccharide building blocks 139–144 (Figure 15). In particular, hydroxyl group at C-4 in place of the labile carbonyl occurring in the native ST5 CPS. Seeberger via an azido-phenylselenation reaction on the double bond. Among the oligomers synthesized, L-fucosamine acceptor 143 the andprotective L-pneumosamine donor 144 were both synthesized frommicroarrayL-fucal 145 via group [89] uncovered ST5 CPS epitope using a combination of glycan oligomer 136a contains N-acetyl-D-quinovosamine (A’, D-QuiNAc) in place of of Sugp (A), displaying an azido-phenylselenation on the doubleevaluation bond. Among the oligomers synthesized, oligomer based mAb generationreaction and immunological performed in rabbit models. These a hydroxyl group at C-4 in place of the labile carbonyl occurring in the native ST5 CPS. Seeberger 136a contains N-acetylD-quinovosamine (A’, D-QuiNAc) in place of of Sugp (A), displaying a hydroxyl group [89] uncovered the protective ST5 CPS epitope using a combination of glycan microarraygroupbased at C-4mAb in place of the labile carbonyl occurring in theperformed native ST5 Seeberger generation and immunological evaluation in CPS. rabbit models. group These [89]
uncovered the protective ST5 CPS epitope using a combination of glycan microarray-based mAb
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generation and immunological evaluation performed in rabbit models. These experiments showed that the rare aminosugar L-PneuNAc, as well as the branching, are essential for antibody recognition Molecules 2018, 23, x FOR PEER REVIEW 17 of 52 and avidity. Interestingly, it was also demonstrated that CRM197 glycoconjugate 136b, containing experiments showed that the rare aminosugar L-PneuNAc, as well ascompared the branching, are essential for 197 D -QuiNAc, induced higher antibody titers and opsonic activity to native ST5-CRM antibody recognition and avidity. Interestingly, it wasshould also demonstrated that CRM197 conjugate contained in PCV13 vaccine. Special care be taken, however, inglycoconjugate the interpretation containing D-QuiNAc, induced highervaccination antibody titers and opsonici.e., activity comparedvaccine to nativevs. a of the136b, results obtained with such different modalities, a 13-valent ST5-CRM 197 conjugate contained in PCV13 vaccine. Special care should be taken, however, in the monovalent synthetic vaccine. The latter indeed contains only one type of carbohydrate which is interpretation of the results obtained with such different vaccination modalities, i.e., a 13-valent administered in much higher amount in comparison to the same carbohydrate contained in PCV13. vaccine vs. a monovalent synthetic vaccine. The latter indeed contains only one type of carbohydrate Nevertheless, this result suggests the possibility for the replacement of labile functional groups, which is administered in much higher amount in comparison to the same carbohydrate contained in generating with stable the functional groups do not affect the functional immunogenic PCV13.manufacture Nevertheless, issues, this result suggests possibility for thethat replacement of labile properties of glycoconjugates. groups, generating manufacture issues, with stable functional groups that do not affect the immunogenic properties of glycoconjugates.
7.6. S. pneumoniae Serotype 8
7.6. S. pneumoniae Serotype 8
ST8 is part of the first-generation polysaccharide vaccine PPV23, but it is not included in ST8 is vaccines part of the first-generation polysaccharide vaccine PPV23, but it iswere not included in glycoconjugate PCV7, PCV13 and PCV10. Many clinical ST8 isolates found resistant glycoconjugate vaccines PCV7,clindamycin, PCV13 and PCV10. Many clinical ST8 isolates were found resistant to this to antibiotics like erythromycin, tetracycline and ciprofloxacin [90]. Furthermore, antibiotics like erythromycin, clindamycin, tetracycline and ciprofloxacin [90]. Furthermore, this multiresistant serotype is a major cause for concern in HIV-infected patients, where its occurrence multiresistant serotype is a major cause for concern in HIV-infected patients, where its occurrence is is significantly more frequent [91]. ST8 CPS consists of a linear tetrasaccharide repeating unit significantly more frequent [91]. ST8 CPS consists of a linear tetrasaccharide repeating unit (Figure (Figure 16) sharing a common cellobiuronic acid disaccharide [β-D-GlcA-(1→4)-β-D-Glc] with ST3 16) sharing a common cellobiuronic acid disaccharide [β-D-GlcA-(1→4)-β-D-Glc] with ST3 CPS (BA CPS (BA sequence, sequence, FigureFigure 12). 12).
Figure 16. Repeating unit of Streptococcus pneumoniae 8 CPS and synthetic ST8 glycan fragments
Figure 16. Repeating unit of Streptococcus pneumoniae 8 CPS and synthetic ST8 glycan fragments reported in [92]. reported in [92].
Schumann et al. [92] reported the preparation of tetrasaccharide fragments of ST8 CPS 146a– 150a, to identify protective glycan epitope. The four tetrasaccharides Schumann et al.the [92]minimal reported the preparation of tetrasaccharide fragments ofwere ST8synthesized CPS 146a–150a, by automated glycan protective assembly, using solid-phase synthesis, starting from building by to identify the minimal glycan epitope.oligosaccharide The four tetrasaccharides were synthesized blocks 151–156. Glycan microarray containing all ST8 CPS frameshifts led to the identification one automated glycan assembly, using solid-phase oligosaccharide synthesis, starting fromofbuilding tetrasaccharide frameshift (BAEC, 148a) that was preferentially recognized by a protective mAb, a blocks 151–156. Glycan microarray containing all ST8 CPS frameshifts led to the identification of murine immunoglobulin M (IgM) against native ST8 CPS [93]. Conjugation with CRM197 of the one tetrasaccharide frameshift (BAEC, 148a) that was preferentially recognized by a protective mAb, tetrasaccharide 148a gave the ST8 glycoconjugate 148b, which was used for immunization of mice a murine immunoglobulin M (IgM) against native ST8acid CPS [93]. Conjugation CRM 197 of the and rabbit models. Interestingly, although cellobiuronic disaccharide conjugate with 103b (BA, Figure tetrasaccharide 148a gave the ST8 glycoconjugate 148b, which was used for immunization of mice and 12) conferred protective immunity against ST3 [81], no ST3-directed immune response was found rabbitafter models. Interestingly,with although cellobiuronic acid disaccharide 103bpresentation (BA, Figure 12) mice immunization conjugate 148b (BAEC), probably becauseconjugate of the different of cellobiuronic in the ST8 sequence. with CRM197immune and coformulation with found PCV13 after conferred protectiveacid immunity against ST3Conjugation [81], no ST3-directed response was
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mice immunization with conjugate 148b (BAEC), probably because of the different presentation of cellobiuronic acid in the ST8 sequence. Conjugation with CRM197 and coformulation with PCV13 of 2018, 23, x FOR PEER of 52 eitherMolecules tetrasaccharide 148a orREVIEW its congener tetrasaccharide 150a (AAEC), containing a D-Glc18residue in place of the naturally occurring D-GlcA, led to a new vaccine which conferred protective immunity in of either tetrasaccharide 148a or its congener tetrasaccharide 150a (AAEC), containing a D-Glc residue rabbits against all the 14 S. pneumoniae serotypes. This achievement confirms the possibility of adding in place of the naturally occurring D-GlcA, led to a new vaccine which conferred protective immunity synthetic oligosaccharide antigens to existing vaccines with the aim of expanding current formulations in rabbits against all the 14 S. pneumoniae serotypes. This achievement confirms the possibility of and replacing serotypes that are not antigens efficiently adding synthetic oligosaccharide to targeted. existing vaccines with the aim of expanding current formulations and replacing serotypes that are not efficiently targeted.
7.7. S. pneumoniae Serotypes 14 and 19F
7.7. S. pneumoniae Serotypes 14 and 19F Among the synthetic glycoconjugate vaccines for S. pneumoniae, gold nanoclusters have been Among thehighlighting synthetic glycoconjugate vaccines for S.carriers pneumoniae, nanoclusters have been recently explored, the potential of these for gold the development of synthetic recently explored, highlighting the potential of these carriers for the development of synthetic vaccines [94,95]. In particular, glyconanoparticles bearing the synthetic tetrasaccharide epitope of vaccines [94,95]. In particular, bearing the synthetic tetrasaccharide epitope of S. S. pneumoniae type 14 (ST14) PSglyconanoparticles have been recently reported [96]. ST14 PS consists of repeating pneumoniae type 14 (ST14) PS have been recently reported [96]. ST14 PS consists of repeating units of units of the tetrasaccharide (6)-[β-D-Galp-(1→4)-]β-D-GlcpNAc-(1→3)β-D-Galp-(1→4)β-D-Glcp-(1→)n the tetrasaccharide (6)-[β-D-Galp-(1→4)-]β-D-GlcpNAc-(1→3)β-D-Galp-(1→4)β-D-Glcp-(1→)n (Figure (Figure 17) [97]. 17) [97].
P
Figure 17. Repeating unit of S. pneumoniae type 14 (ST14) and S. pneumoniae type 19F (ST19F), and
Figure 17. Repeating unit of S. pneumoniae type 14 (ST14) and S. pneumoniae type 19F (ST19F), and their their synthetic fragment conjugated to gold nanoparticles reported in Reference [96] and in Reference synthetic [98]. fragment conjugated to gold nanoparticles reported in Reference [96] and in Reference [98].
The synthetic branched tetrasaccharideGal-Glc-(Gal-)GlcNAc Gal-Glc-(Gal-)GlcNAc (157a), synthesized and and studied The synthetic branched tetrasaccharide (157a), synthesized studied by Mawas et al. [99] was identified as the smallest structure producing protective antibodies by Mawas et al. [99] was identified as the smallest structure producing protective against antibodies ST14 when conjugated to CRM197 protein (glycoconjugate 157b) [100]. Tetrasaccharide 157c, against ST14 when conjugated to CRM197 protein (glycoconjugate 157b) [100]. Tetrasaccharide 157c, derivatized with a terminal thiol for nanoparticle functionalization, was conjugated together with the derivatized with a terminal thiol for nanoparticle functionalization, was conjugated together with the T cell-stimulating ovalbumin peptide (OVA 323–329) and D-glucose fragment 158 to produce small T cell-stimulating peptide (OVA and D-glucose fragment 158 to produce (2 nm) hybridovalbumin gold glyconanoparticles 159323–329) (GNPs, Figure 17). Immunogenicity studies in micesmall (2 nm) hybridthat gold 159 (GNPs, Figure 17). Immunogenicity mice showed showed 159glyconanoparticles induced the production of specific IgG antibodies against ST14 studies PS. The in presence of that 159 the production of specific IgG antibodies againstIgG ST14 PS. Thewhile presence of OVA OVAinduced 323–339 peptide was necessary for the induction of high affinity antibodies, the T cell epitope did was not necessary raise anti-OVA peptide antibodies, thusantibodies, avoiding the riskthe ofTepitope 323–339 peptide for the323–339 induction of high affinity IgG while cell epitope suppression. Sera obtained from mice immunized with 159 with a ratio of tetrasaccharide:Glc:OVA did not raise anti-OVA 323–339 peptide antibodies, thus avoiding the risk of epitope suppression. Sera 323–339 = 45:50:5 were able to with opsonize although less efficiently than sera from mice obtained from mice immunized 159 ST14 withbacteria, a ratio of tetrasaccharide:Glc:OVA 323–339 = 45:50:5 immunized with native ST14 PS conjugated to CRM197. These results make 159 a promising S. were able to opsonize ST14 bacteria, although less efficiently than sera from mice immunized with pneumoniae type 14 vaccine candidate. native ST14 PS conjugated to CRM197 . These results make 159 a promising S. pneumoniae type 14 In another recent study [98], gold glyco-nanoparticles (GNP) were prepared with synthetic vaccine candidate. oligosaccharide fragments corresponding to the repeating units of S. pneumoniae CPS type 19F and In recent study [98], gold glyco-nanoparticles (GNP)unit were prepared with synthetic 14. another In particular trisaccharide 140a, corresponding to ST19F repeating [β-D -ManpNAc-(1→4)-αoligosaccharide fragments corresponding to the repeating units of S. pneumoniae CPS type D-Glcp-(1→2)-α-L-Rhap-(1→] (Figure 17), was prepared according to procedures described in 19F the and 14. Inliterature particular trisaccharide 140a, corresponding ST19F160b. repeating unit [β-D157c -ManpNAc-(1 [101,102] and derivatized as thiol-endingtoligand Tetrasaccharide (fragment → of 4)-αST14), 160b Pn19F), D-glucose according fragment 158 OVA 323–339 peptide D -Glcp-(1 →trisaccharide 2)-α-L-Rhap-(1 →(fragment ] (Figure of 17), was prepared to and procedures described in the were loaded onto GNPs (161, Figure 17) in different ratios. After mice immunization, GNPs 161
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literature [101,102] and derivatized as thiol-ending ligand 160b. Tetrasaccharide 157c (fragment of ST14), trisaccharide 160b (fragment of Pn19F), D-glucose fragment 158 and OVA 323–339 peptide were loaded onto GNPs (161, Figure 17) in different ratios. After mice immunization, GNPs 161 enhanced the production of specific IgG antibodies toward ST14 PS, while no IgG antibodies against ST19F PS were elicited. InMolecules particular, of specific IgG antibodies towards ST14 polysaccharide raised by 161 2018, 23, the x FORtiters PEER REVIEW 19 of 52 were higher than the titers elicited by GNPs exclusively displaying ST14 (159), and comparable with enhanced the production of specific IgG antibodies toward ST14 PS, while no IgG antibodies against commercially available PCV13. Of note, this work explored the effect on the immunological response ST19F PS were elicited. In particular, the titers of specific IgG antibodies towards ST14 polysaccharide of glyconanoparticles displaying twothe carbohydrate epitopes from different bacterial serotypes. raised by 161 were higher than titers elicited by GNPs exclusively displaying ST14 (159), and comparable with commercially available PCV13. Of note, this work explored the effect on the
8. Group Aimmunological Streptococcus response of glyconanoparticles displaying two carbohydrate epitopes from different bacterial serotypes.
Group A Streptococcus (GAS) is a Gram-positive microorganism causing post-sequelae autoimmune 8. Group A Streptococcus infections including rheumatic heart disease. The main driver of autoimmunity is the surface-anchored GAS M polymorphic [103]. Indeed, M protein-based vaccines were Group A proteins Streptococcus (GAS) is a formulated Gram-positivemultivalent microorganism causing post-sequelae autoimmune heart disease. The main driver autoimmunity is the tested in animal andinfections humanincluding modelsrheumatic but they are protective only forof the serotypes included in surface-anchored GAS M polymorphic proteins [103]. Indeed, formulated multivalent M proteinformulation. For this reason, the identification of a common protective antigen is highly desirable. based vaccines were tested in animal and human models but they are protective only for the Due to itsserotypes prominence the GAS For cellthiswall and its conservation across all GAS strains, the includedin in formulation. reason, the identification of a common protective antigen Lancefieldisgroup A carbohydrate (GAC) hasinbeen considered asits a potential a universal highly desirable. Due to its prominence the GAS cell wall and conservationantigen across allfor GAS strains, the Lancefield group A carbohydrate (GAC) has been considered as a potential for a GAS vaccine [103]. The Lancefield group A carbohydrate (GAC) consists of aantigen α-L-Rhap(1 →3)-αuniversal GAS vaccine [103]. The Lancefield group A carbohydrate (GAC) consists of a α-LL -Rhap(1→2)-[β- D -GlcpNAc]-(1→3) repeating unit (Figure 18). Rhap(1→3)-α-L-Rhap(1→2)-[β-D-GlcpNAc]-(1→3) repeating unit (Figure 18).
Figure 18. Repeating unit of the cell wall PS of GAS and synthetic fragments reported in [104].
Figure 18. Repeating unit of the cell wall PS of GAS and synthetic fragments reported in [104]. Increasing concerns regarding autoreactivity of antibodies that recognize the native GAC GlcNAc side chain (anti-GlcNAc monoclonal antibodies) [105], however, have been supported by Increasing concerns regarding autoreactivity of antibodies that recognize the native GAC GlcNAc recent studies [106,107]. Cross-reactivity (especially in heart or brain tissues) of anti-GlcNAc mAb side chainwas (anti-GlcNAc monoclonal antibodies) [105], however, have been supported hypothesized almost twenty years ago [108] and it is still a crucial point of discussion, as well as by recent the role of polyrhamnose on the(especially immunogenicity. More recently, Henningham et al. [109] reportedmAb was studies [106,107]. Cross-reactivity in heart or brain tissues) of anti-GlcNAc that the relativetwenty contribution of GlcNAc side chains thestill innate GAS varies as well hypothesized almost years ago [108] and itto is a immune crucial resistance point ofofdiscussion, among strains and that GlcNAc side chain is not a universal GAS virulence factor in animal models. as the role of polyrhamnose on the immunogenicity. More recently, Henningham et al. [109] In 2010, Kabanova et al. [104] reported the synthesis of two sets of hexasaccharide- and reported that the relative contribution GlcNAc(Figure side 18) chains to the innate immune resistance of dodecasaccharide-CRM 197 conjugates of 162b–165b and compared their immunogenicity with the native GAC-CRM conjugate. Allside oligomers synthesized starting from factor in GAS varies among strains and 197 that GlcNAc chain162a–165a is not awere universal GAS virulence buildingIn blocks 166Kabanova and 167. Of et note, GACreported isolated from fermentation was found to be animal models. 2010, al.the [104] thebacterial synthesis of two sets of hexasaccharidecontaminated with polyrhamnose variant species. The synthetic oligosaccharide conjugates 162b– and dodecasaccharide-CRM 162b–165b (Figure 18) and compared their immunogenicity 197 conjugates 165b showed similar immune response in mice compared to GAC conjugate against two GAS isolates with the native GAC-CRM conjugate. All length oligomers 162a–165a were starting from 197 A saccharide chain of M1 and M23 serotypes. of six (the minimal size of thesynthesized antigen) was found to be sufficient to elicit building blocks 166 and 167. protective Of note,antibodies. the GAC isolated from bacterial fermentation was found to be
contaminated with polyrhamnose variant species. The synthetic oligosaccharide conjugates 162b–165b showed similar immune response in mice compared to GAC conjugate against two GAS isolates of M1
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and M23 serotypes. A saccharide chain length of six (the minimal size of the antigen) was found to be sufficient to elicit protective antibodies. More recently, Auzanneau et al. [110] reported the synthesis of hexasaccharide 164a and its conjugation to TT carrier protein to give 164c. Previously reported conformational analysis had shown that the branch-point in the trisaccharide repeating motif was important for antibody recognition in the Molecules 2018, 23, x FOR PEER REVIEW 20 of 52 antibody-ligand complex [111]. Epitope mapping of a branched trisaccharide and a doubly-branched hexasaccharide by saturation transfer difference NMR methods [112] confirmed the importance of the More recently, Auzanneau et al. [110] reported the synthesis of hexasaccharide 164a and its branched trisaccharide that was studied bindingreported experiments with a mouse conjugation to TT epitope carrier protein to give 164c. in Previously conformational analysismonoclonal had antibody. The that immunogenicity of the synthetic hexasaccharide–TT 164cfor was confirmed by shown the branch-point in the trisaccharide repeating motif conjugate was important antibody the antibody-ligand complex [111]. Epitope mapping of a branched trisaccharide primaryrecognition (IgM) andinsecondary antibody (IgG) responses, with anti-hexasaccharide titers thatand increased a doubly-branched hexasaccharide by saturation transfer difference NMR methods [112] confirmed after booster immunizations to mice. These titers were similar to those obtained with the native theconjugate. importance of the branched trisaccharide epitope that was studied in binding experiments with GAC–TT a mouse monoclonal antibody. The immunogenicity of the synthetic hexasaccharide–TT conjugate 164c was confirmed by primary (IgM) and secondary antibody (IgG) responses, with anti9. Group B Streptococcus hexasaccharide titers that increased after booster immunizations to mice. These titers were similar to those obtained with theor native GAC–TT conjugate. (GBS) is the leading cause of invasive infections Streptococcus agalactiae Group B Streptococcus
in pregnant women [113], newborns, and elderly people, resulting in pneumonia, sepsis and 9. Group B Streptococcus meningitis [114,115]. GBS is a multiserotype Gram-positive bacterium that expresses Lancefield Streptococcus agalactiae or Group B Streptococcus the leading cause of invasive infections group B polysaccharide as a major virulence factor.(GBS) Ten isdifferent serotypes of GBS PS have been in pregnant women [113], newborns, and elderly people, resulting in pneumonia, sepsis and characterized (Ia, Ib, II, III, IV, V, VI, VII, VIII, IX), but five of them (Ia, Ib, II, III and V) account meningitis [114,115]. GBS is a multiserotype Gram-positive bacterium that expresses Lancefield for the vast of the disease [116]. Chemical of the repeating unit some groupmajority B polysaccharide as a major virulence factor.synthesis Ten different serotypes of GBS PS of have beenof these serotypes (types Ia, II, V) have been reported in recent years by Guo and Gao groups [117–119]. characterized (Ia, Ib, II, III, IV, V, VI, VII, VIII, IX), but five of them (Ia, Ib, II, III and V) account for These works be ofuseful for the synthesis other offragments of unit GBSof PS and for further the vast may majority the disease [116]. Chemical of synthesis the repeating some of these serotypeslike (types Ia, II, V) have been reported in recentstudies. years byGBS Guo and Gao [117–119]. investigations, antigenicity and immunological type IIIgroups repeating unit These is composed works may be useful for the synthesis of other fragments of GBS PS and for further investigations, of →4-β-D-Glcp-(1→6)-β-D-GlcpNAc-[α-NeuNAc-(2→3)-β-D-Galp-β-(1→4)]-(1→3)-β-D-Galp-(1→ like antigenicity and immunological studies. GBS type III repeating unit is composed of →4-β-D-Glcp(Figure 19). (1→6)-β-D-GlcpNAc-[α-NeuNAc-(2→3)-β-D-Galp-β-(1→4)]-(1→3)-β-D-Galp-(1→ (Figure 19).
Figure 19. GBS PSIII repeating unit.
Figure 19. GBS PSIII repeating unit. Baker et al. [113,120] reported that GBS PSIII conjugated to TT carrier protein resulted in high tolerance administered to pregnant women, raising highlyto specific IgG Abs titers which were in high Baker et al.when [113,120] reported that GBS PSIII conjugated TT carrier protein resulted transferred through the placenta to infants. Chemical synthesis of fragments of PSIII and of related tolerance when administered to pregnant women, raising highly specific IgG Abs titers which were desialylated fragments have been reported [121–124]. Conformational studies and molecular transferred through the placenta toshowed infants.high Chemical of fragments of related dynamics simulations [125,126] flexibilitysynthesis of GBS PSIII, as it adoptsofa PSIII partialand helical desialylated fragments have reported [121–124]. Conformational studies and molecular dynamics conformation thanks to been the presence of α-NeuNAc-(2→3)-βD-Galp-β-(1→4) branch, while without simulations [125,126] showed high flexibility of GBSis PSIII, as itInadopts a partial helical conformation the sialic acid residues a random coil conformation preferred. particular, it has been shown that there a specificofinteraction between the sialic acid residues and the while glucosyl and galactosyl thanks to theispresence α-NeuNAc-(2 →3)-βD -Galp-β-(1 →4) branch, without the sialic acid backbone which influences the orientation of the side chain and the backbone conformation [125,126]. residues a random coil conformation is preferred. In particular, it has been shown that there is a These behaviors have been rationalized hypothesizing the existence of an extended conformational specific interaction between the sialic acid residues and the glucosyl and galactosyl backbone which epitope. A recent study carried out by Adamo et al. [127] showed that synthetic fragments of GBS influences the orientation of the side chain and the backbone conformation [125,126]. These behaviors PSIII conjugated to CRM 197 are recognized by polyclonal PSIII specific serum and that the presence have been rationalized hypothesizing existence of an extended conformational epitope. of the branch is a structural relevantthe motif for the recognition of anti-PSIII antibodies. Even if theseA recent neo-glycoconjugates can’t et still considered vaccine promising way study carried out by Adamo al.be[127] showed thatcandidates, synthetic this fragments ofresult GBSpaves PSIIIthe conjugated to to the use of synthetic GBS PSIII oligosaccharide as tools to study their detailed molecular interactions CRM197 are recognized by polyclonal PSIII specific serum and that the presence of the branch is a with anti-PSIII mAbs.
structural relevant motif for the recognition of anti-PSIII antibodies. Even if these neo-glycoconjugates can’t still be considered vaccine candidates, this promising result paves the way to the use of synthetic GBS PSIII oligosaccharide as tools to study their detailed molecular interactions with anti-PSIII mAbs.
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Salmonella enterica serovar Typhi, generally termed Salmonella Typhi (S. Typhi) is a highly invasive encapsulated Gram-negative bacterium, responsible for typhoid fever, a systemic infection mostly 10. Salmonella Typhi diffused in less-developed geographic areas, lacking proper sanitary Infections Salmonella enterica serovar Typhi, generally termed Salmonella Typhi (S.conditions. Typhi) is a highly invasiveoccur generally via consumption of contaminated food and water. Global estimates of typhoid fever burden encapsulated Gram-negative bacterium, responsible for typhoid fever, a systemic infection mostly rangediffused between 11 and 21 million cases and approximately 128,000 to 161,000 deaths annually in less-developed geographic areas, lacking proper sanitary conditions. Infections occur[128], with agenerally peak incidence in individuals from early 15 years old [129]. Clinical via consumption of contaminated foodchildhood and water. to Global estimates of typhoid feverdiagnosis burden of range between 11 and 21 to million casesnon-specific and approximately 128,000 161,000fever deaths annually the infection is difficult due the often symptoms of to typhoid that can be[128], confused with acommon peak incidence individuals from to 15 tests yearsthat old [129]. diagnosis of and with other febrileinillnesses [130] andearly duechildhood to serological oftenClinical give false-negative the infection is difficult due to often non-specific symptoms of typhoid that canisbe confused false-positive results [131,132]. S. the typhi capsule contains three antigens: thefever H antigen a heat sensitive with other common febrile illnesses [130] and due to serological tests that often give false-negative protein of the peritrichous flagellae, while the O or somatic antigen is a cell-wall lipopolysaccharide. and false-positive results [131,132]. S. typhi capsule contains three antigens: the H antigen is a heat The Vi antigen is the capsular polysaccharide which overlies the O antigen. The Vi antigen plays a sensitive protein of the peritrichous flagellae, while the O or somatic antigen is a cell-wall crucial role in the modulation of early inflammatory responses during S. Typhi lipopolysaccharide. The Vi antigen is the capsular polysaccharide which overlies theinfections O antigen.[133,134] The and represents the basis for the formulation of vaccines against this bacterium [134–136]. is called Vi antigen plays a crucial role in the modulation of early inflammatory responses during S. ItTyphi Vi (“Virulence”) antigen due to its ability to for enhance S. Typhi virulence is an anionic infections [133,134] and represents the basis the formulation of vaccines[137,138]. against thisItbacterium [134–136]. It is called Vi → (“Virulence”) antigen due to its ability to enhance S. Typhi virulence [137,138]. polymer composed by α-(1 4)-linked N-acetyl galactosaminuronic acid repeating units predominantly It is an anionic polymer composed by α-(1→4)-linked galactosaminuronic acid repeating O-acetylated at position 3 (Figure 20). The degree ofN-acetyl 3-O-acetylation ranges from 60% to more units predominantly O-acetylated at position 3 (Figure 20). The degree of 3-O-acetylation ranges from of than 90% in some strains and the immunogenicity of Vi antigen is closely related to its degree 60% to more than 90% in some strains and the immunogenicity of Vi antigen is closely related to its and O-acetylation [139]. The carboxylic acids are less exposed and partially shielded by the O-acetyls degree of O-acetylation [139]. The carboxylic acids are less exposed and partially shielded by the Othis can explain the minor effect upon the immunological properties observed after reduction of the acetyls and this can explain the minor effect upon the immunological properties observed after carboxylic acids [139]. reduction of the carboxylic acids [139].
Figure 20. Repeating unit of S. Typhi Vi CPS and synthetic fragments reported in [140–142].
Figure 20. Repeating unit of S. Typhi Vi CPS and synthetic fragments reported in [140–142].
Although pure polysaccharide vaccines based on the purified Vi antigen have been proven effective in pure adults,polysaccharide they have been vaccines so far ineffective in infants (especially children younger thanproven 5 Although based on the purified Vi antigen have been years age), in thehave elderly andsoimmunocompromised individuals [143]. The coupling of S. Typhi effective inof adults, they been far ineffective in infants (especially children younger than 5 years CPSin fragments to carrier (rEPA, TT, CRM197individuals ) produced glycoconjugate vaccines of ableS.toTyphi elicit CPS of age), the elderly and proteins immunocompromised [143]. The coupling a T cell dependent immune response [144,145]. In particular, the conjugate vaccine Vi-TT was recently fragments to carrier proteins (rEPA, TT, CRM197 ) produced glycoconjugate vaccines able to elicit a T found effective in the prevention of typhoid fever in a phase 2b trial and proven to be safe and highly cell dependent immune response [144,145]. In particular, the conjugate vaccine Vi-TT was recently found effective in the prevention of typhoid fever in a phase 2b trial and proven to be safe and
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highly immunogenic [146]. The injectable Vi-TT conjugate vaccine (TCV) is currently licensed and Molecules 2018, 23, x FOR PEER REVIEW 22 of 52 recommended by WHO for children from 6 months of age and adults up to 45 years of age. Synthetic oligomers of Vi antigen reported in the literaturevaccine by Sinaÿ and coworkers In particular, immunogenic [146].were Thefirst injectable Vi-TT conjugate (TCV) is currently[140]. licensed and Vi oligosaccharides up to hexasaccharide 168–172 (Figure 20) bearing an unnatural O-methyl group recommended by WHO for children from 6 months of age and adults up to 45 years of age. Synthetic both at the C4 position of the upstream residue and at C-1 position of the downstream residue have oligomers of Vi antigen were first reported in the literature by Sinaÿ and coworkers [140]. Inbeen particular, Vi precluding oligosaccharides up to hexasaccharide 168–172 (Figure 20) bearing an unnatural Osynthesized, thus protein conjugation [140]. Recently, Ye and co-workers [141] reported methyl group at the C4173–176 positionas of methyl the upstream residuecontaining and at C-1 position of the downstream the synthesis of Viboth oligomers glycosides an unnatural acetyl group at have been synthesized, thus protein conjugation [140]. Recently, Ye andglycosyl coC-4 ofresidue the upstream residue (Figure 20).precluding In particular, N-acetyloxazolidinone-containing workers [141] reported the synthesis of Vi oligomers 173–176 as methyl glycosides containing an donor 177 and acceptor 178 were used to direct alpha stereoselectivity during glycosylation reactions. unnatural acetyl group at C-4 of the upstream residue (Figure 20). In particular, NELISA competitive assays showed that synthetic tri- and tetra-saccharides 174 and 175 had improved acetyloxazolidinone-containing glycosyl donor 177 and acceptor 178 were used to direct alpha antigenic activities in comparison to Sinaÿ fragments [140]. The authors speculated that improved stereoselectivity during glycosylation reactions. ELISA competitive assays showed that synthetic triaffinities could be ascribed the175 presence of an acetyl group at C-4 of the upstream residue in place and tetra-saccharides 174to and had improved antigenic activities in comparison to Sinaÿ fragments of the[140]. methyl ether present in Sinaÿ’s structures. More recently, Ye et al. [142] synthesized a series The authors speculated that improved affinities could be ascribed to the presence of an acetyl of Vi pseudo-oligosaccharides 179–183 (Figure 20) by carbon spacers through olefin group at C-4 of the upstream residue in place of conjugated the methyl ether present chain in Sinaÿ’s structures. More Ye or et by al. the [142] synthesized moiety a seriesthrough of Vi pseudo-oligosaccharides (Figure 20) cross recently, metathesis 1,2,3-triazole Huisgen cycloaddition179–183 reaction. The binding conjugated by antibodies carbon chain through olefin cross metathesis or by theshowing 1,2,3-triazole affinities to anti-Vi ofspacers proposed mimics 179–183 were investigated, that moiety the affinity through Huisgen cycloaddition reaction. The binding affinities to anti-Vi antibodies of of divalent compounds was generally comparable to the monovalents of the same length.proposed For example, mimics 179–183 were investigated, showing that the affinity of divalent compounds was generally the affinity of 181, containing the butylene linker did not increase significantly when compared with comparable to the monovalents of the same length. For example, the affinity of 181, containing the that of monovalent tetrasaccharide. Heterodimer 182, which mimics the native Vi antigen with the butylene linker did not increase significantly when compared with that of monovalent similar chain-elongation direction did not result in improved antigenicity, perhaps suggesting that tetrasaccharide. Heterodimer 182, which mimics the native Vi antigen with the similar chainlongerelongation Vi oligomers are needed higher affinity. antigenicity, perhaps suggesting that longer Vi direction did notfor result in improved Recently, di- and trisaccharide fragments of S. Typhi Vi capsular polysaccharide oligomersthe are synthesis needed forof higher affinity. analoguesRecently, and theirthe zwitterionic has beenfragments accomplished by the group (Figure 21) [147]. synthesis ofcounterparts di- and trisaccharide of S. Typhi Vi Lay capsular polysaccharide analogues and their zwitterionic counterparts has been accomplished by the Lay group (Figure 21) These fragments were composed of N-acetylgalactosaminuronic acid repeating units non-acetylated [147]. These fragments were composed of N-acetylgalactosaminuronic acid repeating units at position 3 (Figure 21). The synthetic strategy was designed in order to obtain the twonondistinct at position 3 (Figure 21). Thederivatives synthetic strategy waszwitterionic designed in order to obtain thecommon two seriesacetylated of oligomers 184–187 (2-acetamido and their analogues) from distinct series of oligomers 184–187 (2-acetamido derivatives and their zwitterionic analogues) from building blocks, donor 188 and glycosyl acceptor 189, in turn synthesized from commercially available common building blocks, donor 188 and glycosyl acceptor 189, in turn synthesized from D-galactosamine hydrochloride. Glycosylation reaction of acceptor 189 with donor 188 gave exclusively commercially available D-galactosamine hydrochloride. Glycosylation reaction of acceptor 189 with the desired α (1,4) disaccharide and the same 1,2-cis stereoselective outcome was observed for donor 188 gave exclusively the desired α (1,4) disaccharide and the same 1,2-cis stereoselective the trisaccharides. outcome was observed for the trisaccharides.
Figure 21. Retrosynthetic analysis of di- and trisaccharide fragments of S. Typhi Vi CPS, reported in
Figure 21. Retrosynthetic analysis of di- and trisaccharide fragments of S. Typhi Vi CPS, reported in Reference [147]. Reference [147].
ELISA tests showed that oligosaccharides 184–187 were recognized by specific anti-Vi polyclonal in a oligosaccharides concentration-dependent with similarby efficacies, than the ELISA testsantibodies showed that 184–187manner were recognized specificlower anti-Vi polyclonal natural Vi polysaccharide. This might be related to the short chain length of the synthetic fragments antibodies in a concentration-dependent manner with similar efficacies, lower than the natural Vi and to the lack of the 3-O-acetyl group, which has been reported as being important for the polysaccharide. This might be related to the short chain length of the synthetic fragments and to the immunogenicity [139].
lack of the 3-O-acetyl group, which has been reported as being important for the immunogenicity [139].
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11. Pseudomonas aeruginosa 11. Pseudomonas aeruginosa Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that can cause hospitalhospital-associated infections, often life-threatening in critically ill patients [148]. Cystic fibrosis associated infections, often life-threatening in critically ill patients [148]. Cystic fibrosis patients often patients often become infected with P. aeruginosa in chronic lung infections [149]. P. aeruginosa encodes become infected with P. aeruginosa in chronic lung infections [149]. P. aeruginosa encodes several several multidrug efflux pump genes [150] and has acquired multiple resistance mechanisms to most multidrug efflux pump genes [150] and has acquired multiple resistance mechanisms to most antibiotic classes, selected by years of antibiotic treatments [151]. In the past few years, new approaches antibiotic classes, selected by years of antibiotic treatments. [151] In the past few years, new such as the administration of anti-bacterial monoclonal antibodies are being for the approaches such as the administration of anti-bacterial monoclonal antibodies areinvestigated being investigated prevention or treatment of P. aeruginosa infections [152]. In a recent study, P. aeruginosa bloodstream for the prevention or treatment of P. aeruginosa infections. [152]. In a recent study, P. aeruginosa infection isolates from patients withpatients acute P.with aeruginosa were analyzed for the ability bloodstream infection isolates from acute P. infections aeruginosa infections were analyzed for the to express a type 3PcrV, secretion protein, and Pslprotein, exopolysaccharide, an important component of the abilityPcrV, to express a type 3 secretion and Psl exopolysaccharide, an important microbial biofilm extracellular matrix [153]. The study showed that the majority of isolates expresses component of the microbial biofilm extracellular matrix [153]. The study showed that the majority of PcrV and Psl. However, mostPsl. of However, the patient’s lacked IgG and active responses isolates expresses PcrV and mostsera of the patient’s sera functionally lacked IgG and functionally to active these targets. findings that Pslsuggest can shield the can bacterium from the host immune responsesThese to these targets.suggest These findings that Psl shield the bacterium from the host immune response, allowing of the bacterium [153]. InPsl particular, Psl is a serotyperesponse, allowing the survival ofthe thesurvival bacterium [153]. In particular, is a serotype-independent independent antigen to in thea helical cell surface in aan helical pattern, that an organization that be antigen anchored to the anchored cell surface pattern, organization can be crucial forcan cell–cell crucial forand cell–cell interactions and to engage in interaction with other components biofilm-initiating components interactions to engage in interaction with other biofilm-initiating [154,155]. [154,155]. Di Giandomenico et al. [156] reported the identification of mAbs, classified in class I, II, and III Di Giandomenico al. [156] epitopes reported of thePsl, identification of mAbs, in class I, II, and III antibodies, binding threeetdifferent as suggested using classified competition antibody binding antibodies, binding three different epitopes of Psl, as suggested using competition antibody binding assays. The mAbs possessed opsonophagocytic killing activity and anti–cell attachment activity. The mAbs possessed opsonophagocytic killing activity and anti–cell attachment activity. In In assays. particular, class I mAb were shown to be the most functionally active and protective anti-Psl particular, class I mAb were shown to be the most functionally active and protective anti-Psl antibodies against P. aeruginosa [156]. The repeating unit of PsI of P. aeruginosa is the pentasaccharide antibodies against P. aeruginosa [156]. The repeating unit of PsI of P. aeruginosa is the pentasaccharide shown in Figure 22, as determined by Kocharova et al. [157]. The chemical synthesis of different shown in Figure 22, as determined by Kocharova et al. [157]. The chemical synthesis of different fragments of PsI, tetra-, penta-, hexa- and decasaccharide 190a–193a was reported starting from fragments of PsI, tetra-, penta-, hexa- and decasaccharide 190a–193a was reported starting from building blocks 194–199 (Figure 22) [158]. building blocks 194–199 (Figure 22) [158].
Figure 22. Repeating unit of PsI of P. aeruginosa and synthetic fragments reported in Reference [158]. Figure 22. Repeating unit of PsI of P. aeruginosa and synthetic fragments reported in Reference [158].
The synthetic strategy dealt with the stereoselective glycosylation of mannosides and the The synthetic strategy dealt with stereoselective glycosylation ofand mannosides and the formation of two 1,2-cis mannosides, onethe of which is also extended at C-1, C-2, C-3 in a crowded formation of two 1,2-cisInmannosides, one of which protected is also extended C-1, [159] C-2, 194 andand C-3195, in a 1,2,3-cis configuration. particular, 4,6-O-benzylidene mannosylatdonors crowded 1,2,3-cis particular, protected mannosyl donors [159] modified by a configuration. C-3 Nap etherInand a C-2 4,6-O-benzylidene silyl ether, respectively, provided optimal 1,2-cis 194stereoselectivity and 195, modified Nap ether and a C-2 silyl respectively, provided optimal in by thea C-3 glycosylation reactions. On ether, the other hand, mannosyl donor 1,2-cis 196, functionalizedinwith participatingreactions. acetyl ester C-2, resulted fordonor the preparation of 1,2stereoselectivity the aglycosylation Onatthe other hand,suitable mannosyl 196, functionalized trans mannosides.acetyl Compounds 190a–193a were used for to the identify the epitope requirements of with a participating ester at C-2, resulted suitable preparation of 1,2-trans mannosides. monoclonal antibodies of class I, II, and III, showing some new insights about immune recognition Compounds 190a–193a were used to identify the epitope requirements of monoclonal antibodies P. aeruginosa [158]. Oligosaccharides 190a–193a were conjugated to BSAPsl of of class I, II, and Psl III, exopolysaccharide showing some new insights about immune recognition of P. aeruginosa
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exopolysaccharide [158]. Oligosaccharides 190a–193a were conjugated to BSA (190b–193b) to facilitate coating of ELISA plates followed by testing reactivity with an antibody that bound each epitope class. The class II mAb reacted potently with all oligosaccharides, suggesting that the epitope for this class resides within tetrasaccharide 190b and does not require the 1,2-cis mannoside of compound 191b. The class III antibody did not bind tetra- (190b) or pentasaccharide BSA-conjugate (191b). On the contrary, it showed weak affinity to glycoconjugate 193b and strong affinity to glycoconjugate 192b, suggesting that the terminal glucoside contained in glycoconjugate 192b is required for optimal binding. The class I antibody did not bind any of the oligosaccharides, suggesting the possibility that the class I mAb binds to a conformational epitope of PsI or to a substructure yet to be determined. The identification of this epitope could provide an attractive lead compound for the development of a synthetic Psl-based vaccine for P. aeruginosa. 12. Neisseria meningitidis Neisseria meningitidis is a Gram-negative bacterium that colonizes the mucous membranes of humans. Meningococcal meningitis and sepsis are severe diseases that kill children and young adults within hours despite the availability of effective antibiotics. Mortality rates and permanent disability, like amputation, hearing loss and neurologic deficiency associated with N. meningitidis infections are high, even in countries where optimal health care practices are in place [160]. Among the 12 serogroups of N. meningitidis [161], serogroups B, C, Y and W cause approximately 90% of invasive meningococcal infections. Group A, however, is the only meningococcal serotype capable of causing of meningitis epidemics. Serogroups B and C express α-(2,8)- and α-(2,9)-linked polysialic acid, respectively. Alternating sequences of D-galactose or D-glucose and sialic acid are expressed by serogroups W and Y [162,163]. The serogroup A capsule is composed of α-(1,6)-linked N-acetyl-D-mannosamine-1-phosphate repeating units, partially acetylated at 3-OH (about 70%) and 4-OH (10–30%) [164]. First generation polysaccharide-based vaccines against N. meningitidis comprise the bivalent (groups A and C), the trivalent (groups A, C and W), and the tetravalent (groups A, C, Y and W) forms. Among second generation meningococcal glycoconjugate vaccines, three monovalent group C conjugate vaccines and one tetravalent meningococcal conjugate vaccine against groups A, C, Y and W are currently available. Of note, serogroup B (Men B) is not included in current formulations and remains a major cause of endemic meningitis in both developed and developing countries. The main obstacle for group B polysaccharides vaccine development is that the group B polysaccharide, composed of α-(2,8)-sialic acid polymers, is expressed in a number of human neurologic tissues since early fetal development. Men B CPS is therefore perceived as self-antigen by the innate immune system and it induces immune tolerance. Structural modification of this “self” antigen replacing the N-acetyl group of sialic acid units with an N-propanonyl group [165] induced high levels of bactericidal IgG antibodies without detection of autoantibodies [166]. However, its development has been suspended due to the poor performance of the vaccine in a limited human trial and to the high perceived risk of autoimmunity [61]. A new type of group B vaccine, Bexsero® (GlaxoSmithKline) developed by conjugation of three recombinant surface antigens (PorA, NadA and fHbp) and outer membrane vesicles from group B strain NZ98/254, is now licensed in more than 35 countries worldwide, including the EU, Australia, Brazil, Canada, Chile, Uruguay and the USA [167–169]. More recently, a new anti-MenB vaccine based on two recombinant lipidated factor H binding protein (Trumenba® , Pfizer) has been licensed by FDA and approved for use in EU countries in 2017. 12.1. N. meningitidis Serogroup A (MenA) N. meningitidis serogroup A is most often implicated in seasonal epidemic diseases, especially in sub-Saharan Africa and asian developing countries [170,171]. The MenA CPS structure consists of (1→6)-linked 2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, with about 70% of O-acetylation at 3-OH (Figure 23).
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P
Figure 23. Men A CPS carbocyclic analogues reported in Reference [172].
Figure 23. Men A CPS carbocyclic analogues reported in Reference [172].
The synthesis of MenA CPS fragments was reported in 2002 by Pozgay [173] and by Oscarson in 2005 [174], and conjugation to HSA was the synthetic fragments found to be immunogenic. The synthesis ofupon MenA CPS fragments reported in 2002 were by Pozgay [173] and by Oscarson MenA CPS, suffers from in water, due to were the chemical of the in 2005 [174], andhowever, upon conjugation to poor HSA stability the synthetic fragments found tolability be immunogenic. phosphodiester linkages involving the anomeric position of each repeating unit. This issue stimulated MenA CPS, however, suffers from poor stability in water, due to the chemical lability of the the design of novel and hydrolytically stable analogues of MenA CPS repeating unit, like carbocyclic phosphodiester linkages involving the anomeric position of each repeating unit. This issue stimulated analogues (Figure 23) [172] and 1-C-phosphono analogues (Figure 24) [175,176], where a methylene the design of novel and hydrolytically stable analogues of MenA CPS repeating unit, like carbocyclic group replaces the pyranose oxygen atom or the anomeric oxygen, respectively. The conformational analogues (Figure 23) [172] and 1-C-phosphono analogues (Figure 24) [175,176], where a methylene behaviour of these analogues was investigated through DFT calculation and NMR spectroscopy group replaces pyranose oxygen or the of anomeric oxygen, The conformational [177], with athe particular focus on theatom orientation the phosphate or respectively. phosphonate aglycone and on behaviour of these analogues investigated through calculation and NMR spectroscopy the possibility of pyranosewas ring inversion [178]. TheDFT comparison between mimics and natural[177], with fragment a particular focus the orientation the phosphate or phosphonate and of on the showed the on preservation of the 4Cof 1 geometry in both classes of analogues.aglycone The synthesis carbocyclic stabilizedring analogues of MenA wasbetween reported mimics by the Lay group withfragment the possibility of pyranose inversion [178].CPS Thefragments comparison and natural 4 C dimer obtainment of monomer 200a, 201a and trimer 202a of carba-N-acetylmannosamine-1-Oshowed the preservation of the 1 geometry in both classes of analogues. The synthesis of carbocyclic phosphate. The formation of the phosphodiester wasby achieved use of obtainment the Hstabilized analogues of MenA CPS fragments wasbridges reported the Laythrough group the with the phosphonate methodology [179], followed by functionalization with a phosphodiester-linked of monomer 200a, dimer 201a and trimer 202a of carba-N-acetylmannosamine-1-O-phosphate. aminopropyl spacer to allow protein conjugation. Oligomer synthesis was achieved starting from The formation of the phosphodiester bridges was achieved through the use of the H-phosphonate carbasugar 203, derived from compound 204. Carbocycle formation was carried out by Claisen methodology [179],offollowed byinfunctionalization a phosphodiester-linked aminopropyl spacer rearrangement glucal 205, turn obtained fromwith commercially available glucal 206 [172,177]. The to allow proteinabilities conjugation. Oligomermolecules synthesiswere was achieved starting carbasugar derived inhibition of the synthetic investigated by a from competitive ELISA203, assay, fromshowing compound 204. Carbocycle formation was carried out by Claisen rearrangement glucal 205, that carba-disaccharide 201a is recognized by a polyclonal anti-MenA serum with anofaffinity in turn obtained from commercially available glucal 206 polymerization [172,177]. Thedegree inhibition similar to a native MenA oligosaccharide with average of 3 abilities [172]. Theof the conjugation of carbocyclic analogues 200a–202a to the protein carrier CRM 197 gave glycoconjugates synthetic molecules were investigated by a competitive ELISA assay, showing that carba-disaccharide (Figure that were tested for immunogenicity MenA fragments, 201a 200b–202b is recognized by23) a polyclonal anti-MenA serum with[180]. an affinity similar toproduced a nativeby MenA mild acid hydrolysis of native MenA polysaccharide (average degree of polymerization from to 15) oligosaccharide with average polymerization degree of 3 [172]. The conjugation of 6carbocyclic and conjugated to CRM197 were used to compare the activity of 200b–202b. Upon mice immunization, analogues 200a–202a to the protein carrier CRM197 gave glycoconjugates 200b–202b (Figure 23) that all glycoconjugates elicited antibodies that recognized the respective structures, although only were tested for immunogenicity [180]. MenA fragments, produced by mild acid hydrolysis of native conjugated trimer 202b was able to induce specific anti-MenA IgG antibodies with detectable in vitro MenA polysaccharide degree polymerization from 6 to andextent conjugated to CRM197 bactericidal activity.(average Compound 202b, of however, elicited antibodies to a15) lesser than hexamer wereand used to compare the activity of 200b–202b. Upon mice immunization, all glycoconjugates pentadecamer conjugated oligomers 207 and 208 obtained from hydrolysis of the native elicited antibodies that recognized the respective structures, although polysaccharide, suggesting that hydrolytically stable analogues of MenAonly CPS conjugated can be used trimer for the 202b was able to induce specific anti-MenA IgG antibodies with detectable in vitro bactericidal activity. Compound 202b, however, elicited antibodies to a lesser extent than hexamer and pentadecamer conjugated oligomers 207 and 208 obtained from hydrolysis of the native polysaccharide, suggesting
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that hydrolytically stable analogues of MenA CPS can be used for the development of vaccine and that conjugates with longer carbocyclic oligomers could further increase the induced immune response. Molecules 2018, 23, x FOR PEER REVIEW 26 of 52 In addition, a strategy for the multivalent presentation of carba analogues was developed [181] allowing conjugation of monomer and dimer 201a to the metallic surface of superparamagnetic development of vaccine and that 200a conjugates with longer carbocyclic oligomers could further increase iron oxide nanoparticles toIn generate 200c and 201c 23). SPIONs can act asofmultivalent the induced immune(SPION) response. addition, a strategy for(Figure the multivalent presentation carba analogues developed [181] conjugation of monomer 200a and dimer 201a to the metallic carriers and as was a contrast agent forallowing magnetic resonance imaging (MRI) [182]. Functionalized SPIONs surface superparamagnetic iron oxideinto nanoparticles (SPION) atoreduction generate 200c 201c (Figure dispersed in of aqueous media can aggregate clusters inducing of T2and [183] and this event 23). SPIONs can as multivalent carriers and a contrast agentMR for image magnetic resonanceThis imaging can be monitored asact a decrease in brightness of as a T2-weighted [183,184]. property (MRI) [182]. Functionalized SPIONs dispersed in aqueous media can aggregate into clusters inducing has been widely used for ligand detection in biological media [185]. SPIONs 200c and 201c were a reduction of T2 [183] and this event can be monitored as a decrease in brightness of a T2-weighted produced as approximately spherical nanoparticles, with a size dispersion of 13 ± 3 nm and an average MR image [183,184]. This property has been widely used for ligand detection in biological media particle coating of 200c 320 unities per nanoparticle forapproximately 200c and of 160 ligands per nanoparticle for 201c, [185]. SPIONs and 201c were produced as spherical nanoparticles, with a size as determined by transmission electron microscopy (TEM). Both 200c and 201c were able to bind dispersion of 13 ± 3 nm and an average particle coating of 320 unities per nanoparticle for 200c and the polyclonal anti-MenA antibody, as by MRI analysis, exploiting themicroscopy magnetic (TEM). peculiarity of 160 ligands per nanoparticle forevaluated 201c, as determined by transmission electron Both 200c and 201c were able to bind the polyclonal anti-MenA antibody, as evaluated by MRI of SPIONs. analysis, exploiting the magnetic peculiarity The synthesis of C-phosphono analogues of ofSPIONs. N. meningitidis group A CPS oligomers was reported The synthesis of C-phosphono analogues of meningitidis A CPS oligomers wasthe reported by the Oscarson [176] and Lay [186] groups. In N. particular, angroup improved strategy for synthesis by the Oscarson [176] and Lay [186] groups. In particular, an improved strategy for the synthesis of of monosaccharides 209a–210a and phosphonoester-bridged fragments 211a–213a was recently monosaccharides 209a–210a and phosphonoester-bridged fragments 211a–213a was recently reported [175] starting from compound 214, 215 and 216. The introduction of the phosphonate reported [175] starting from compound 214, 215 and 216. The introduction of the phosphonate moiety moiety was accomplished on alcohol 217, obtained from α-C-allenyl derivative 218 which, in turn, was was accomplished on alcohol 217, obtained from α-C-allenyl derivative 218 which, in turn, was prepared in sixinsteps from orthoester intermediate 24). prepared six steps from orthoester intermediate 219 219 (Figure (Figure 24).
Figure 24. Retrosynthetic strategy for the synthesis of C-phosphono analogues of MenA CPS reported
Figure 24. Retrosynthetic strategy for the synthesis of C-phosphono analogues of MenA CPS reported in [174] and multivalent presentation of the fragments [181]. in [174] and multivalent presentation of the fragments [181].
Competitive ELISA assay showed that monosaccharides 209a–210a and synthetic fragments 211a–213a containing unnatural phosphonoester linkage were 209a–210a recognized by a human polyclonal Competitive ELISAthe assay showed that monosaccharides and synthetic fragments anti-MenA serum [175]. The comparison with the inhibition of either MenA (positive control) or 211a–213a containing the unnatural phosphonoester linkage were recognized by a human polyclonal MenY (negative control) indicated that the chain lengths of the saccharide molecules is important for anti-MenA serum [175]. The comparison with the inhibition of either MenA (positive control) or the efficacy, while the presence of the phosphonate residue (comparison between compounds 211a– MenY (negative control) indicated that the chain lengths of the saccharide molecules is important 213a and glycosides 209a–210a) and the orientation of the anomeric linker (comparison between for the efficacy, while the presence of the phosphonate residue (comparison between compounds compounds 211a and 212a) did not affect the affinity. Multivalent presentation on gold nanoparticles 211a–213a and glycosides 209a–210a) and the orientation of the(GNPs anomeric linker between of monomer 209a, dimer 211a and trimer 213a were obtained 220, 221 and(comparison 222 respectively, compounds 211a andInterestingly, 212a) did not affect the affinity. presentation on gold Figure 24) [187]. nanoparticles 220, 221Multivalent and 222 showed a more than threenanoparticles order of of monomer 209a, dimer 211aaffinity and trimer 213acounterparts were obtained (GNPsto220, 221 and 222 respectively, magnitude higher binding than their not bound the gold cluster 209a, 211a and 213a, at the same nominal concentration of saccharides. Fallarini et al. [188] used functionalized Figure 24) [187]. Interestingly, nanoparticles 220, 221 and 222 showed a more than three order of gold nanoparticles to affinity test their ability induce immune cell responses as cluster a consequence of and magnitude higher binding than theirtocounterparts not bound to the gold 209a, 211a
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213a, at the same nominal concentration of saccharides. Fallarini et al. [188] used functionalized gold nanoparticles to test their ability to induce immune cell responses as a consequence of multivalency. In particular, monodisperse gold nanoparticles (2 and 5 nm) coated with mono- and disaccharides Molecules 23, xsynthesized. FOR PEER REVIEW 27 of 52 the (220 and 221)2018, were Conjugation to gold nanoparticles conferred to the saccharides ability to activate macrophages and this property is dependent on the size of the nanoparticles, with multivalency. In particular, monodisperse gold nanoparticles (2 and 5 nm) coated with mono- and 5 nm disaccharides nanoparticles giving comparable results toConjugation those obtained with the polysaccharide (220 and 221) were synthesized. to gold nanoparticles conferred bacterium to the capsule (MenA) used as a natural antigen. Activation of macrophages occurred, independently saccharides the ability to activate macrophages and this property is dependent on the size of the of the saccharide oligomerization (or charge) on the nanoparticle surface. only nanoparticles nanoparticles, with 5 nm nanoparticles giving comparable results to However, those obtained with the 220, exposing a phosphonodisaccharide-functionalized monolayer, induced T cells polysaccharide bacterium capsule (MenA) used as a natural antigen. Activation of proliferation macrophages and occurred,ofindependently of the saccharide (or charge) on marker the nanoparticle the increase released interleukin-2 levels,oligomerization the latter being a typical of T cellsurface. activation. However, only nanoparticles 220, exposing a phosphonodisaccharide-functionalized monolayer, Recently, HSA conjugates 209b, 211b and 213b [189] were shown to induce both T cell proliferation induced T cellsrelease proliferation andand the to increase of released interleukin-2 the latterproduction being a typical and interleukin-2 in vitro, stimulate moderate specific levels, IgG antibody in vivo. marker of T cell activation. Recently, HSA conjugates 209b, 211b and 213b [189] were shown to induce All HSA-conjugated compounds 209b, 211b and 213b induced T cell proliferation (40% of proliferation both T cell proliferation and interleukin-2 release in vitro, and to stimulate moderate specific IgG at 102 µM), whereas only phosphonodisaccharide 211a was effective (28% of proliferation at 102 µM) antibody production in vivo. All HSA-conjugated compounds 209b, 211b and 213b induced T cell among the unconjugated showing behavior of phosphonodisaccharide triggering T cell proliferation in vitro proliferation (40% offorms, proliferation at the 102 unusual μM), whereas only 211a was and causing release. at 102 μM) among the unconjugated forms, showing the unusual effectiveinterleukin-2 (28% of proliferation behavior of triggering T cell proliferation in vitro and causing interleukin-2 release.
12.2. N. meningitidis Serogroup C (MenC)
12.2. N. meningitidis Serogroup C (MenC) N. meningitidis group C CSP is a α-(2,9)-polysialic acid with sporadic 7/8-O-acetylation N. meningitidis group C fragments CSP is a α-(2,9)-polysialic acid with 7/8-O-acetylation (Figure (Figure 25). Non-acetylated have been shown to sporadic be immunogenic and to elicit an 25). Non-acetylated fragments have been shown to be immunogenic and to elicit an immune response immune response that is effective in recognizing and killing the bacterium [190]. A series of that is effective in recognizing and of killing the bacterium [190]. A series non-acetylated α-2,9non-acetylated α-2,9-oligosialic acids different length 223a–233a wereofprepared by a convergent oligosialic acids of different length 223a–233a were prepared by a convergent synthetic route synthetic route employing 5N,4O-oxazolidinone-protected phosphate-based building blocks 234 and employing 5N,4O-oxazolidinone-protected phosphate-based building blocks 234 and acceptor 235 acceptor 235 [191]. The dodecamer was synthesized with a [4+8] strategy that allowed to retain the [191]. The dodecamer was synthesized with a [4+8] strategy that allowed to retain the α-selectivity α-selectivity even sizeand of donor and acceptor increased. even when thewhen size ofthe donor acceptor increased.
Figure 25. Repeating unit of MenC CPS and synthetic fragments reported in [191,192].
Figure 25. Repeating unit of MenC CPS and synthetic fragments reported in [191,192].
In a further study, di-, tri-, tetra-, and penta-sialic acids 223a–226a were coupled with KLH
In a further study, tri-, tetra-, and penta-sialic acids were coupled with KLH carrier carrier protein and di-, studied for immunogenicity [192]. The223a–226a glycoconjugates elicited robust T cellmediated immunity mice, in particular[192]. the immunogenicity of the tested oligo sialic acids increased protein and studied forinimmunogenicity The glycoconjugates elicited robust T cell-mediated in the in order tri(224b) > di(223b) tetra(225b) > penta(226b). The antibodies elicited were tested forin the immunity mice, in particular the> immunogenicity of the tested oligo sialic acids increased efficacy and specificity, verifying if they could recognize and target group C N. meningitidis. Allefficacy of order tri(224b) > di(223b) > tetra(225b) > penta(226b). The antibodies elicited were tested for the antisera obtained with the oligosaccharide-conjugates 223b–226b had strong binding to the N. and specificity, verifying if they could recognize and target group C N. meningitidis. All of the antisera meningitidis cell and no significant binding to cells not expressing α-2,9-poly and oligosialic acids obtained with the oligosaccharide-conjugates 223b–226b had strong binding to the N. meningitidis cell (sialoglycans sTn, GM3, GM2, α-2,8-linked polysialic). The α-2,9-trisialic acid was identified as a and no significant binding to cells notglycoconjugate expressing α-2,9-poly and oligosialic acids (sialoglycans promising antigen for developing vaccines against group C Neisseria meningitidis. sTn, GM3,Recently, GM2, α-2,8-linked polysialic). The α-2,9-trisialic identified aswere a promising antigen α-2,9-oligosialic acid fragments (di-, tri-, tetra-,acid andwas penta, 223a–226a) conjugated with for developing glycoconjugate vaccines against group C Neisseria meningitidis. Recently, α-2,9-oligosialic monophosphoryl lipid A (MPLA), a known immune potentiator (molecule with adjuvant activity) acid fragments (di-, tri-, tetra-, penta, 223a–226a) were (Figure conjugated [193]. Immunological studiesand of the conjugates 223c–226c 25) inwith micemonophosphoryl revealed that they lipid elicited arobust immune responses, mainly IgG2b and IgG2c, consistent with[193]. T cellImmunological dependent A (MPLA), known immune potentiator (molecule with adjuvant activity) immunities. In particular, the immune response was comparable to the corresponding KLH-
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studies of the conjugates 223c–226c (Figure 25) in mice revealed that they elicited robust immune responses, mainly IgG2b and IgG2c, consistent with T cell dependent immunities. In particular, the immune response was comparable to the corresponding KLH-conjugates 223b–226b plus adjuvant. The immunogenicity of oligosialic acids decreased with elongated sugar chain, although all tested MPLA conjugates exhibited strong immune responses. The dimer 223c, actually, induced the highest 2018, 23, x FOR total PEER REVIEW 28 of 52did not titers ofMolecules antigen-specific and IgG2b antibodies, but some of the produced antibodies bind to oligosialic acids or bacterial CPS. In contrast, the tri- and tetra-sialic acid−MPLA conjugates conjugates 223b–226b plus adjuvant. The immunogenicity of oligosialic acids decreased with 224c–225c, were sugar fully chain, effective showing theMPLA highest binding to bacterial andresponses. were identified as elongated although all tested conjugates exhibited strongcells immune The promising vaccine candidates worthy of further dimer 223c, actually, induced the highest titersinvestigation. of antigen-specific total and IgG2b antibodies, but some of the produced antibodies did not bind to oligosialic acids or bacterial CPS. In contrast, the tri-
12.3. N. and meningitidis Wconjugates (MenW) 224c–225c, were fully effective showing the highest binding tetra-sialicSerogroup acid−MPLA to bacterial cells and were identified as promising vaccine candidates worthy of further investigation.
N. meningitidis serogroup W CPS consists of a glycan repeating unit of [→6)-α-D-Galp-(1→4)-αD -Neup5Ac(7/9OAc)-(2 ] (Figure 26). The synthesis of MenW CPS oligosaccharides of various 12.3. N. meningitidis→ Serogroup W (MenW) lengths was N. performed by Wu group in 2013 [194], the aim of unit studying theDrelationship between meningitidis serogroup W CPS consists of awith glycan repeating of [→6)-α-Galp-(1→4)-αoligosaccharide length and immunogenicity. D-Neup5Ac(7/9OAc)-(2→] (Figure 26). The synthesis of MenW CPS oligosaccharides of various lengths was by Wu group in 2013 [194], withobtained the aim ofstarting studyingfrom the relationship Oligomers upperformed to decasaccharides 236a–240a were protectedbetween disaccharides oligosaccharide length and immunogenicity. 241 and 242, bearing a N-acetyl-5-N,4-O-oxazolidinone (Figure 26), in turn synthesized from sialyl Oligomers up to decasaccharides 236a–240a were obtained starting from protected disaccharides phosphate donor 243, functionalized with an oxazolidinone and a phosphate leaving group to increase 241 and 242, bearing a N-acetyl-5-N,4-O-oxazolidinone (Figure 26), in turn synthesized from sialyl the α-selectivity, and galactosides 244 and 245. Compound 243 was prepared from compound 246. phosphate donor 243, functionalized with an oxazolidinone and a phosphate leaving group to Oligosaccharide elongation wasand accomplished by iterative increase the α-selectivity, galactosides 244 and 245. glycosylation Compound 243 and was deprotections, prepared from using disaccharide 241 for theOligosaccharide [2+n] glycosylation reactions. After conjugation with CRM compound 246. elongation was accomplished by iterative glycosylation and 197 , immunization deprotections, usinganalysis disaccharide 241 for theantisera [2+n] glycosylation reactions. Afterinduced conjugation of mice and glycan array of produced showed that antibodies by with conjugated CRM197 , immunization of mice array analysis produced showed thatlonger antibodies disaccharide 236b recognized onlyand the glycan disaccharide itself of and did notantisera cross react with oligomers. induced by conjugated disaccharide 236b recognized only the disaccharide itself and did not cross In contrast, antibodies induced by glycoconjugates 237b, 238b, 239b and 240b all recognized tetra- to react with longer oligomers. In contrast, antibodies induced by glycoconjugates 237b, 238b, 239b and decasaccharides, but not disaccharides. A serumbut bactericidal assay A was used to studyassay the bactericidal 240b all recognized tetra- to decasaccharides, not disaccharides. serum bactericidal was activity used of thetoantibodies and showed that of 236b not induce antibodies withdid bactericidal study the bactericidal activity the did antibodies and showed that 236b not induceactivity. antibodies longer with bactericidal activity. thein contrary, longer oligomers could and in particular, the with On the contrary, oligomers couldOn and particular, the tetramer 239b elicited antibodies tetramer 239b elicited antibodies with the highest bactericidal effect. Taken together, these data the highest bactericidal effect. Taken together, these data suggested that the tetrasaccharide is the suggested that the tetrasaccharide is the minimum saccharide length required to induce bactericidal minimum saccharide length required to induce bactericidal antibodies. antibodies.
Figure 26. Repeating unit of MenW CPS and synthetic strategy used the construction of MenW CPS
Figure 26. Repeating unit of MenW CPS and synthetic strategy used the construction of MenW CPS fragments [194]. fragments [194].
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12.4. N. meningitidis Serogroup X (MenX) 12.4. N. meningitidis Serogroup X (MenX) Serotype X of N. meningitidis (MenX) emerged as a substantial threat to public health, especially in Serotype X of N. meningitidis (MenX) emerged as a substantial threat to public health, especially the “meningitis belt” area, after the introduction of MenA vaccine (MenAfriVac) and other conjugate in the “meningitis belt” area, after the introduction of MenA vaccine (MenAfriVac) and other vaccines (consisting of MenA, C, Y and W serotypes) that do not provide coverage for MenX conjugate vaccines (consisting of MenA, C, Y and W serotypes) that do not provide coverage for serotype [195,196]. As a consequence, there is the need to extend the protection of current vaccines, MenX serotype [195,196]. As a consequence, there is the need to extend the protection of current developing more comprehensive conjugate vaccines [197]. The MenX CPS is a homopolymer of vaccines, developing more comprehensive conjugate vaccines [197]. The MenX CPS is a 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate moiety (Figure 27) [198,199]. homopolymer of 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate moiety (Figure 27) [198,199]. In 2013 Morelli et al. reported the synthesis of monomer 247a, dimer 248a and trimer 249a of In 2013 Morelli et al. reported the synthesis of monomer 247a, dimer 248a and trimer 249a of N. N. meningitidis X CPS [200], starting from intermediates 250–252, in turn derived from D-GlcNAc. meningitidis X CPS [200], starting from intermediates 250–252, in turn derived from D-GlcNAc. P
P
P
P
P
P
P
P
P
P
P
P
P
Figure 27. Repeating unit of MenX CPS and synthetic fragments reported in [200–202]. Figure 27. Repeating unit of MenX CPS and synthetic fragments reported in [200–202].
In 2014 an improvement of the synthesis of MenX fragments, their conjugation to CRM197 and In 2014 an improvement MenX fragments, their to CRM and 197mice the immunological evaluationof ofthe thesynthesis resulting of conjugates 247b–249b wasconjugation reported [203]. Upon the immunological evaluation of the resulting conjugates 247b–249b was reported [203]. Upon immunization, the conjugated trimer (249b) was found as the minimal fragment possessing mice immunization, the conjugated trimer (249b)lower was found the minimal fragment possessing immunogenic activity, although significantly than as pentadecasaccharide-conjugate 253 immunogenic activity, although significantly lower than pentadecasaccharide-conjugate 253 obtained obtained from the native polymer and used in the same study. This finding suggests that oligomers from native polymer and used the same study. This finding suggests that oligomers longerthethan three repeating unitsinwere possibly needed to mimic the activity of the longer native than three repeating units wereyear, possibly needed mimic an thealternative activity of synthesis the nativeofpolysaccharide. polysaccharide. The following Harale et al. to reported the tetrameric The following year, Harale al. reported synthesis the tetramericend, fragment of MenX fragment of MenX CPS et 254a, lacking an thealternative phosphate at the ofdownstream starting from CPS 254a, lacking the phosphate at the downstream end, starting from intermediates 251 and in intermediates 251 and 225 in turn synthesized from monosaccharide 256 (Figure 27) 225 [201]. turn synthesized fromexperiment, monosaccharide (Figure 27) [201]. ELISA using Competitive ELISA using 256 MenX bacterial CPSCompetitive as a control, gaveexperiment, a concentrationMenX bacterial CPS as a control, gave a concentration-dependent inhibition of anti-MenX antibodies dependent inhibition of anti-MenX antibodies from both compound 254a (unconjugated synthetic from compound 254a (unconjugated synthetic MenXinhibition tetramer) for andunconjugated tetramer-TT conjugate MenXboth tetramer) and tetramer-TT conjugate 254b. Lower fragment 254b. 254a Lower inhibition for unconjugated fragment 254a (up to 68% inhibition) was observed compared (up to 68% inhibition) was observed compared to its conjugate form 254b (up to 89% inhibition) to at its form 254b (up to 89% inhibition) all antigen concentrations tested. Also, bacterial all conjugate antigen concentrations tested. Also, bacterialat MenX CPS showed higher inhibition than synthetic MenX CPS showed higher inhibition than synthetic compounds at all an respective concentrations compounds at all respective concentrations used [201]. Very recently, alternative strategy for used [201]. Very recently, an alternative strategy for MenX oligomer synthesis (Figure was MenX oligomer synthesis (Figure 27) was developed [202], based on an enzyme-catalyzed 27) one-pot developed [202], based on an enzyme-catalyzed one-pot elongation of synthetic trimer 257a. Oligomers elongation of synthetic trimer 257a. Oligomers with predefined average length (compound 258a for with predefined (compound 258a for general formula avDP 12) were synthesized general formula average of avDPlength = 12) were synthesized and conjugated to of CRM 197. = Mice immunized with and conjugated to CRM . Mice immunized with 258b elicited functional antibodies comparable to 258b elicited functional197antibodies comparable to controls immunized with the current MenX controls immunized with the current MenX glycoconjugates prepared from the naturalproduced. CPS or from its glycoconjugates prepared from the natural CPS or from its fragments enzymatically fragments enzymatically produced. 13. Mycobacterium tuberculosis Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the leading causes of death in the world. In 2016, some 1.7 million people died from the disease, with 64% cases of the
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13. Mycobacterium tuberculosis Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the leading causes of death in the world. In 2016, some 1.7 million people died from the disease, with 64% cases of the total occurring in India, Indonesia, China, Philippines, Pakistan, Nigeria, and South Africa [204]. Moreover, it is estimated that one-third of the human population is latently infected with M. tuberculosis Molecules 2018, 23, x FOR PEER REVIEW of 52 to TB). and is highly vulnerable if immunocompromised (in 2016, 40% of HIV deaths were30due The mycobacterial cell wall is a highly complex structure largely composed of carbohydrates and total occurring in India, Indonesia, China, Philippines, Pakistan, Nigeria, and South Africa [204]. lipids. Major components are lipidated polysaccharides, like the mycolyl−arabinogalactan complex, Moreover, it is estimated that one-third of the human population is latently infected with M. essentially composed galactofuranose (Galf ) and arabinofuranose ) [205]. recent tuberculosis and isof highly vulnerable if immunocompromised (in 2016, 40%(Araf of HIV deaths In were due years, numerous efforts have been done toisdevelop inhibitors of UDP-galactopyranose mutase (UGM), to TB). The mycobacterial cell wall a highly complex structure largely composed of carbohydrates and lipids. Major areand lipidated polysaccharides, like the mycolyl−arabinogalactan an enzyme essential forcomponents the growth survival of this mycobacterium [206–212]. Among the essentially composed of galactofuranose (Galf)mannosides and arabinofuranose [205]. In recent vital cellcomplex, envelope components, phosphatidylinositol (PIMs)(Araf) and their corresponding years, numerous efforts have been done to develop inhibitors of UDP-galactopyranose mutase hyper-mannosylated derivatives (lipomannans and lipoarabinomannans) are noncovalently anchored (UGM), an enzyme essential for the growth and survival of this mycobacterium [206–212]. Among to the plasma membrane and the outer capsule via their lipid chains. PIMs have a crucial role in the vital cell envelope components, phosphatidylinositol mannosides (PIMs) and their corresponding the intracellular life of the bacterium, by binding toand macrophages, to Toll-like and C-type hyper-mannosylated derivatives (lipomannans lipoarabinomannans) arereceptors noncovalently lectins [213,214], antigenand presenting cell surfaces. PIMs also PIMs activate anchored toexpressed the plasma on membrane the outer capsule via their lipid chains. havenatural a crucial killer T the intracellular life of the bacterium, binding to macrophages, to Toll-like receptors and moiety cells forrole theinproduction of interferon-γ [215]. byStructurally, PIMs consist of a myo-inositol C-type lectins [213,214], expressed on antigen presenting cell surfaces. PIMs also activate natural linked with a diacylated glycerophospholipid unit and two α-mannosylation sites at O2 and O6 killer T cells for the production of interferon-γ [215]. Structurally, PIMs consist of a myo-inositol (Figure 28, compounds 259–261). When additional lipid chains are linked to the mannosyl-and moiety linked with a diacylated glycerophospholipid unit and two α-mannosylation sites at O2 and myo-inositol-moieties, triacylated PIMsWhen (AcPIMs) andlipid tetraacylated PIMs are formed. O6 (Figure 28, compounds 259–261). additional chains are linked to (Ac2PIMs) the mannosyl-and Higher myo-inositol-moieties, PIMs (for example, triacylated AcnPIM3PIMs AcnPIM6) are formed by elongation at the mannose (AcPIMs) and tetraacylated PIMs (Ac2PIMs) are formed.residue. Higherof PIMs AcnPIM3 AcnPIM6) are formed by structures elongation at the mannose residue. [216–221]. In In the course the(for pastexample, few years, several synthesis of these have been reported the course of the past few years, several synthesis of these structures have been reported [216–221]. In addition, heterocyclic analogues of PIMs in which the inositol ring is replaced by a piperidine or a In addition,moiety heterocyclic of PIMs in which the inositol ringthe is replaced by aactivity piperidine a parent tetrahydropyran haveanalogues been prepared and shown to retain biological of or the tetrahydropyran moiety have been prepared and shown to retain the biological activity of the parent PIM structures [222]. PIM structures [222].
Figure 28. Phosphatidylinositol mannosides (PIMs) and retrosynthetic route of tetraacylated
Figure phosphatidylinositol 28. Phosphatidylinositol mannosides (PIMs) inand retrosynthetic route of tetraacylated hexamannoside (Ac2PIM6) reported [223]. phosphatidylinositol hexamannoside (Ac2 PIM6 ) reported in [223]. Recently, the synthesis of a tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6, 262) and its immunological evaluation have been reported [223]. Oligomer 262 was synthesized starting Recently, the synthesis of a tetraacylated phosphatidylinositol hexamannoside (Ac2 PIM6 , 262) from pseudotrisaccharide 263, tetramannoside donor 264 and hydrogen phosphonate 265 (Figure 28). and its immunological evaluation reported [223]. Oligomer266, 262267, was synthesized Compounds 263 and 264 were have in turnbeen obtained from monosaccharides 268 and 269, 270,starting respectively through a [1+1] strategy. The immunological evaluation was performed by observing
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from pseudotrisaccharide 263, tetramannoside donor 264 and hydrogen phosphonate 265 (Figure 28). Compounds 263 and 264 were in turn obtained from monosaccharides 266, 267, 268 and 269, 270, respectively through a [1+1] strategy. The immunological evaluation was performed by observing Molecules 2018, 23, x FOR PEER REVIEW 31 of 52 the induction of antigen-specific antibodies in mice immunized with ovalbumin or tetanus toxoid adjuvanted with ofcompound 262. antibodies The adjuvant effects of Alum various PIMs isolated from the induction antigen-specific in mice immunized withorovalbumin or tetanus toxoid M. tuberculosis straincompound H37Rv (iPIM1,2 iPIM6) wereofalso examined inPIMs parallel for comparison. adjuvanted with 262. Theand adjuvant effects Alum or various isolated from M. strain (iPIM1,2 iPIM6) were also examined in parallel for comparison. Miceand Micetuberculosis exposed to the H37Rv synthesized Acand PIM6 262 exhibited increased production of interleukin-4 2 exposed to the synthesized Ac 2 PIM6 262 exhibited increased production of interleukin-4 and interferon-γ, suggesting proper activation of the innate immune system. Interestingly, 262 induced interferon-γ, suggesting proper activation of the innate immune system. Interestingly, 262 induced an approximately two to four-fold increase in the level of antigen specific antibodies, similarly to an approximately four-fold increase the level of antigen specific antibodies, similarly to bacteria-derived PIMstwo andtoslightly lower thaninAlum. bacteria-derived PIMs and slightly lower than Alum. Among M. tuberculosis cell wall polysaccharide and lipid components, some complex structures Among M. tuberculosis cell wall polysaccharide and lipid components, some complex structures of arabinogalactan and lipoarabinomannan (LAM) have been synthesized in recent years [224,225]. of arabinogalactan and lipoarabinomannan (LAM) have been synthesized in recent years [224,225]. In particular, Wang et al. [83] synthesized LAM oligosaccharides 271a–273a (Figure 29), that were In particular, Wang et al. [83] synthesized LAM oligosaccharides 271a–273a (Figure 29), that were conjugated to BSA (271b–273b) and evaluatedwith withimmunological immunological studies. conjugated to BSA (271b–273b) andKLH KLH(271c–273c) (271c–273c) and and evaluated studies.
Figure 29. LAM oligosaccharides reported in Reference [83] and retrosynthetic route to LAM
Figure 29. LAM oligosaccharides reported in Reference [83] and retrosynthetic route to LAM tetrasaccharide coupled to a monophosphoryl lipid A (MPLA), as reported in [226]. tetrasaccharide coupled to a monophosphoryl lipid A (MPLA), as reported in [226].
LAM tetra-, hepta-, and undecasaccharides 271a–273a, containing the α-1,5-, α-1,3-, and β-1,2linked domainand withundecasaccharides the 5-OH of the upstream residues capped with the α-1,2-linked LAM arabinan tetra-, hepta-, 271a–273a, containing the α-1,5-, α-1,3-, and D -arabinose in 10, 15, 14 linear dimannose motif, were synthesized in good overall yields from β-1,2-linked arabinan domain with the 5-OH of the upstream residues capped with and the α-1,2-linked steps, respectively [83]. KLH conjugates 271c–273c and free oligosaccharides 271a–273a were injected dimannose motif, were synthesized in good overall yields from D-arabinose in 10, 15, and 14 linear in mice and the obtained antisera were analyzed by ELISA using BSA conjugates 271b–273b as steps, respectively [83]. KLH conjugates 271c–273c and free oligosaccharides 271a–273a were injected capture antigens. Antisera derived from mice immunized with oligosaccharides 271a–273a did not in mice and the obtained antisera were analyzed by ELISA using BSA conjugates 271b–273b as capture contain carbohydrate antigen-specific antibodies, while all KLH conjugates 271c–273c elicited antigens. Antisera derived from mice immunized with oligosaccharides 271a–273a did not contain antigen-specific immune responses in mice. In particular, antibody titers induced by 271c and 273c carbohydrate antigen-specific antibodies, all KLH conjugates elicited antigen-specific were slightly higher than those inducedwhile by 272c. In a further work271c–273c [226], the LAM tetrasaccharide was coupled to a monophosphoryl lipid A (MPLA) derivative generating a MPLA-based synthetic glycoconjugate 274 (Figure 29). In 274, the carbohydrate antigen was attached to the MPLA C-6′-
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immune responses in mice. In particular, antibody titers induced by 271c and 273c were slightly higher than those induced by 272c. In a further work [226], the LAM tetrasaccharide was coupled to a monophosphoryl lipid A (MPLA) derivative generating a MPLA-based synthetic glycoconjugate 274 (Figure 29). In 274, the carbohydrate antigen was attached to the MPLA C-60 -position via an amide bond. Retrosynthetic analysis of 274 gives LAM tetrasaccharide 275, conveniently synthesized with a [2+2] strategy from 276 and 277 as reported before by the same authors [83], linker 278, and MPLA derivative 279. In turn, 279 was synthesized from fatty acids 280 and 281 and disaccharide 282, Molecules 2018, 23, x FOR PEER REVIEW 32 of 52 assembled from glycosyl donor 283 and acceptor 284. Immunological activity of conjugate 274 was evaluated in mice, IgG antibodyanalysis responses. Since injection position via affording an amide robust bond. Retrosynthetic of 274 givesintraperitoneal LAM tetrasaccharide 275,elicited conveniently synthesized with a [2+2] strategy 276 and 277 injection, as reporteditbefore by the same responses significantly stronger than those fromfrom subcutaneous was hypothesized that authors [83], linker 278, and MPLA derivative 279. In turn, 279 was synthesized from fatty acids 280 MPLA conjugates may stimulate B1 lymphocytes in the intrapleural and peritoneal cavities. These and 281 and disaccharide 282, assembled from glycosyl donor 283 and acceptor 284. Immunological results revealed also the self-adjuvant properties of MPLA conjugates, paving the way to further activity of conjugate 274 was evaluated in mice, affording robust IgG antibody responses. Since investigation of theseinjection compounds as responses antituberculosis vaccine candidates. intraperitoneal elicited significantly stronger than those from subcutaneous injection, it was hypothesized that MPLA conjugates may stimulate B1 lymphocytes in the
14. Fungal Infections intrapleural and peritoneal cavities. These results revealed also the self-adjuvant properties of MPLA
conjugates, paving the way to further investigation of these compounds as antituberculosis vaccine Fungal infections can occur in healthy people, although immunosuppressed or candidates. immunocompromised patients are the major risk group for invasive fungal infections, as well as 14. patients use antibiotics able to modify the human microbiota [227]. Antifungal drugs Fungalwho Infections are commercially available but they are limited compared to antibacterial drugs. In recent years, Fungal infections can occur in healthy people, although immunosuppressed or several strategies have been patients developed for major the identification new anti-fungal compounds, immunocompromised are the risk group forofinvasive fungal infections, as well including as components of who plants, and microorganism [227]. In particular, vaccination patients useanimals antibiotics able to modify the human microbiota [227]. Antifungal offers drugs promising are commercially but they areof limited compared to antibacterial drugs. Intorecent years, several alternative solutionsavailable for the treatment fungal infections that are resistant antibiotics. Recognition strategies have been developed for the identification of new anti-fungal compounds, including of fungi by the innate immune system depends on several Pathogen-Associated Molecular Patterns components of plants, animals and microorganism [227]. In particular, vaccination offers promising (PAMPs) in the fungal cell wall [228]. Specific receptors, exposed on antigen presenting cells surface, alternative solutions for the treatment of fungal infections that are resistant to antibiotics. Recognition are involved of system polysaccharide wall components, likeMolecular the mannose of fungiinbythe therecognition innate immune depends oncell several Pathogen-Associated Patternsreceptor (MR) and DC-SIGN recognition of branched N-linked mannan [229],presenting Toll-likecells receptor 4 (TLR4) (PAMPs) in thefor fungal cell wall [228]. Specific receptors, exposed on antigen surface, are involved in the O-linked recognitionmannan of polysaccharide cell wall3components, like the mannose receptorreceptor for recognition of linear [230], galectin for β-mannosides, complement DC-SIGN for recognition of branched N-linked [229], Toll-like receptor 4 (TLR4) [231]. 3 (CR3)(MR) for and β-(1,6)-glucan, and dectin 1 and TLR2 for mannan β-glucan and phospholipomannan for recognition of linear O-linked mannan [230], galectin 3 for β-mannosides, complement receptor 3 Mannans consist of differently linked oligomannoside, and phospholipomannans are composed of (CR3) for β-(1,6)-glucan, and dectin 1 and TLR2 for β-glucan and phospholipomannan [231]. phospholipids and mannans. The common linkages between mannose units in fungal mannans are Mannans consist of differently linked oligomannoside, and phospholipomannans are composed of α-(1,6), phospholipids α-(1,2)-, α-(1,3) β-(1,2), shownlinkages in Figure 30. β-glucans areincomposed of β-are D -glucose andand mannans. Theas common between mannose units fungal mannans with β-(1,3) linkages and sporadic β-(1,6) branched points. Galactomannans consist of structurally α-(1,6), α-(1,2)-, α-(1,3) and β-(1,2), as shown in Figure 30. β-glucans are composed of β-D-glucose β-(1,3) linkages and sporadic β-(1,6) points. Galactomannans consistto of galactofuranoside structurally diverse with heteropolysaccharides composed of branched a poly-D-mannose backbone linked diverse heteropolysaccharides composed of a polyD -mannose backbone linked to galactofuranoside (Galf ) units (Figure 30). Of note, the galactomannan of Aspergillus fumigatus is a specific carbohydrate (Figure 30). Of note, the galactomannan of Aspergillus fumigatus is a specific carbohydrate antigen (Galf) usedunits for clinical detection of fungal infection. antigen used for clinical detection of fungal infection.
f
f
f
Figure 30. Examples of mannan, β-glucan and galactomannan components of fungal cell walls.
Figure 30. Examples of mannan, β-glucan and galactomannan components of fungal cell walls. In the course of the past few years, several synthetic strategies [232–234] have been developed to prepare these oligosaccharides expressed on the cell wall of various fungi, like Candida albicans and Apergillus fumigatus, as detailed in Sections 14.1 and 14.2.
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In the course of the past few years, several synthetic strategies [232–234] have been developed to prepare these oligosaccharides expressed on the cell wall of various fungi, like Candida albicans and Apergillus fumigatus, detailed in Sections 14.1 and 14.2. Molecules 2018, 23, x FOR as PEER REVIEW 33 of 52 14.1. 14.1. Candida Candida albicans albicans The yeast Candidaalbicans albicans is opportunistic an opportunistic pathogenic microorganism, in the The yeast Candida is an pathogenic microorganism, found found in the normal normal microflora, and the surfaces mucosalofsurfaces of most healthy individuals. However, it is microflora, skin andskin the mucosal most healthy individuals. However, it is able to cause able to cause severe infections in immunocompromised individuals and patients undergoing severe infections in immunocompromised individuals and patients undergoing immunosuppressive immunosuppressive therapy [235]. The three majoron glycans expressed onalbicans the cellare wall of C. albicans are therapy [235]. The three major glycans expressed the cell wall of C. phosphomannanphosphomannan-based glycoproteins in the outermost part of the cell wall and β-glucans and chitin, based glycoproteins in the outermost part of the cell wall and β-glucans and chitin, especially at the especially at the level of the bud scars (Figure 31) [228]. level of the bud scars (Figure 31) [228].
P
Figure 31. Major components of C. Albicans cell wall: β-(1,3)-glucan and chitin (poly-β-(1,4)-NFigure 31. Major components of C. Albicans cell wall: β-(1,3)-glucan and chitin acetylglucosamine) are the main components of inner cell wall. The outer layer is enriched with (poly-β-(1,4)-N-acetylglucosamine) are the main components of inner cell wall. The outer polymannans. layer is enriched with polymannans.
Being part of the phosphomannan glycoproteins, the acid-labile β-mannan is thought to be a part phosphomannan the acid-labile β-mannan is thought to be a majorBeing epitope ofof allthe C. Albicans serotypes glycoproteins, and thus represents an ideal target for vaccine development. major epitope of all C. Albicans serotypes and thus represents an ideal target for vaccine development. On this subject, an extensive work has been carried out by the Bundle group to develop a conjugate On this subject, hasfrom been carried by theofBundle group to develop a conjugate vaccine against an C.extensive albicans, work starting bindingout studies β-mannans with two monoclonal vaccine against C. albicans, starting from binding studies of β-mannans with two monoclonal protective antibodies, the mAb C3.1 and its immunoglobulin M (IgM) counterpart B6.1protective [236]. By antibodies, the mAb C3.1 and its immunoglobulin (IgM) counterpart [236]. By [238], meansthe of means of STD-NMR, computational analysis [237]Mand hydroxyl groupB6.1 replacement STD-NMR, computational analysiselements [237] andwas hydroxyl [238], identification of identification of key recognition used group for thereplacement development of the antigens that elicit key recognition elements was used for the development of antigens that elicit polyclonal protective polyclonal protective antibodies, a process referred to as “reverse engineering” [236]. Initially, a set antibodies, a process referred to aspropyl “reverse engineering” [236].(Figure Initially, set of β-(1-2)-mannan of β-(1-2)-mannan oligosaccharide glycosides 285a–290a 32) aranging in size from dioligosaccharide propyl glycosides 285a–290a (Figure 32) ranging in size from di-to heptasaccharides to heptasaccharides were evaluated against mAbs C3.1 and B6.1. Interestingly, di and trisaccharides were mAbs C3.1binding and B6.1.capacity, Interestingly, and trisaccharides 285a and had 285a evaluated and 286aagainst had maximum whiledilarger oligosaccharides were286a bound maximum binding oligosaccharides bound progressively more weakly [239]. progressively morecapacity, weaklywhile [239].larger This unusual pattern were of inhibition was consistent with a binding This unusual pattern of inhibition was consistent with a binding site that could accommodate the site that could accommodate the trisaccharide, even though the primary polar contacts are located trisaccharide, even though the primary polar contacts are located within the disaccharide. The synthesis within the disaccharide. The synthesis of complementary mono-deoxy and mono-O-methyl of complementary mono-deoxy and mono-O-methyl analogues of dimers 285b, 291b–304b and trimers analogues of dimers 285b, 291b–304b and trimers 286b and 305b (Figure 32) led to epitope mapping 286b and 305b (Figure 32) led to epitope mapping of anti-Candida albicans antibodies [240]. The strategy of anti-Candida albicans antibodies [240]. The strategy for the construction of these β-mannosides is for the construction of these of β-mannosides is based on the formationbyof epimerization β-glucosidic linkages, based on the formation β-glucosidic linkages, followed at C-2followed via an by epimerization at C-2 via an oxidation − reduction sequence. oxidation−reduction sequence. Dimer 285c and trimer 286c (R = (CH2)3S(CH2)2NH2) were conjugated to chicken serum albumin (CSA) and the resulting glycoconjugates 285d and 286d (Figure 32) were able to generate highly specific IgG Abs titers in mice and rabbit [241]. The trisaccharide conjugated to CSA (286d) raised protective antibodies in rabbits [242]. Serum from rabbits immunized by 286d contained antibodies that stained C. albicans cells in fluorescent labeling studies more intensely than antibodies from
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Dimer 285c and trimer 286c (R = (CH2 )3 S(CH2 )2 NH2 ) were conjugated to chicken serum albumin (CSA) and the resulting glycoconjugates 285d and 286d (Figure 32) were able to generate highly specific IgG Abs titers in mice and rabbit [241]. The trisaccharide conjugated to CSA (286d) raised protective antibodies in rabbits [242]. Serum from rabbits immunized by 286d contained antibodies that stained C. albicans cells in fluorescent labeling studies more intensely than antibodies from rabbits Molecules with 2018, 23, x FOR[105,241]. PEER REVIEW of 52 immunized 285d Both glycoconjugates 285d and 286d reduced C. albicans34counts in vital organs but they were insufficient to provide 100% protection. In a further work, trisaccharide–TT counts in vital organs but they were insufficient to provide 100% protection. In a further work, conjugate 286e was found to be a stronger immunogen in rabbits, but it was poorly immunogenic in trisaccharide–TT conjugate 286e was found to be a stronger immunogen in rabbits, but it was poorly mice [241]. However, when the same trisaccharide was conjugated to different T cell peptides, found immunogenic in mice [241]. However, when the same trisaccharide was conjugated to different T cell in cellpeptides, wall proteins expressed during pathogenesis of human candidiasis, the resulting glycopeptides found in cell wall proteins expressed during pathogenesis of human candidiasis, the (286f)resulting elicited aglycopeptides peptide- and(286f) carbohydrate-specific response that gave protection againstthat challenge elicited a peptideand carbohydrate-specific response gave by C. albicans infection in mouse models [243]. All glycoconjugates showed immunogenicity with higher protection against challenge by C. albicans infection in mouse models [243]. All glycoconjugates Abs titers compared to unconjugated 286c. When hyphal wall protein-1 (Hwp1), fructose-bisphosphate showed immunogenicity with higher Abs titers compared to unconjugated 286c. When hyphal wall protein-1 fructose-bisphosphate aldolase (Fba) and methyltetrahydropteroyltriglutamate aldolase (Fba) (Hwp1), and methyltetrahydropteroyltriglutamate (Met6) were used as T cell peptides for (Met6) were used as T rate cell peptides conjugation, the survival rate in mice challenge experiments conjugation, the survival in mice for challenge experiments was 80–100% against 40–80% survival was 80–100% against 40–80% survival with other glycopeptides. with other glycopeptides.
Figure 32. Oligomannans and glycoconjugates synthesized by the Bundle group [236].
Figure 32. Oligomannans and glycoconjugates synthesized by the Bundle group [236].
The C. albicans cell wall consists of approximately 60% β-glucan. Although initially thought to be hidden underneath theconsists mannoprotein layer, recent60% evidence suggest that β-glucans arethought exposedto be The C. albicans cell wall of approximately β-glucan. Although initially on the cell surface, possibly restricted to specific regions, such as bud scars [228]. β-glucans have also hidden underneath the mannoprotein layer, recent evidence suggest that β-glucans are exposed on the been investigated for their binding capacity to dectin-1 [244,245], a dendritic cell receptor thatbeen cell surface, possibly restricted to specific regions, such as bud scars [228]. β-glucans have also mediatesfor phagocytosis and capacity mediator production inflammation caused fungal pathogens investigated their binding to dectin-1during [244,245], a dendritic cell by receptor that mediates [231]. Among β-glucans, Laminarin (Lam) is a polysaccharide extracted from Laminaria digitate plant. phagocytosis and mediator production during inflammation caused by fungal pathogens [231]. Among In 2010, Bromuro et al. [246] investigated the potential of laminarin as antigen for C. albicans, aimed β-glucans, Laminarin (Lam) is a polysaccharide extracted from Laminaria digitate plant. In 2010, at mediating antifungal protection. The authors showed that a synthetic linear structure composed Bromuro et al. [246] investigated therepeating potentialunits of laminarin as antigen for C. albicans, aimed at mediating of pentadecamer of Lam β-(1-3) (15mer) conjugated to CRM 197 conferred protection antifungal protection. The authors showed that a synthetic linear structure composed pentadecamer against C. albicans. In the same study, a synthetic β-(1-6) branched 17mer conjugated toofCRM 197 was of Lam β-(1-3) repeating units (15mer) conjugated to CRM conferred protection against C. albicans. immunogenic but not protective [246], suggesting that 197 the linear β-(1-3) fragment contains the epitope. To further investigate this aspect, Adamo ettoal.CRM [247]197 reported the synthesisbut of not In theprotective same study, a synthetic β-(1-6) branched 17mer conjugated was immunogenic short [246], linear suggesting and β-(1-6) that branched Lam β-(1-3) fragments 306a–309a (Figurethe 33)protective and their epitope. immunological protective the linear fragment contains To further evaluation. investigate this aspect, Adamo et al. [247] reported the synthesis of short linear and β-(1-6) branched Lam fragments 306a–309a (Figure 33) and their immunological evaluation.
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Figure 33. 33. Repeating Repeating unit unit of of Lamarin Lamarin (Lam) (Lam) and and Lam Lam fragments fragments reported reported in in [247]. [247]. Figure
Compounds 306a–315a were synthesized starting from common building blocks 310, 311, 312 Compounds 306a–315a were synthesized starting from common building blocks 310, 311, 312 and 313 (Figure 33). Preliminary ELISA test with fragments 306a–309a showed that compound 309a and 313 (Figure 33). Preliminary ELISA test with fragments 306a–309a showed that compound 309a was the best inhibitor of the binding between Lam and anti-Lam Abs elicited in mice by Lam-CRM197 was the best inhibitor of the binding between Lam and anti-Lam Abs elicited in mice by Lam-CRM197 conjugate, confirming the hypothesis that the linear β-(1-3) fragments contain the dominant epitope. conjugate, confirming the hypothesis that the linear β-(1-3) fragments contain the dominant epitope. Hexasaccharide 314b, prepared from building block 315 (Figure 33), was studied in order to identify Hexasaccharide 314b, prepared from building block 315 (Figure 33), was studied in order to identify the the protective epitope. ELISA assays on compounds 306a–309a and 314b showed that hexasaccharide protective epitope. ELISA assays on compounds 306a–309a and 314b showed that hexasaccharide 314b 314b provided 95% of inhibition at 4 mM concentration, while trisaccharide 309a and tetrasaccharide provided 95% of inhibition at 4 mM concentration, while trisaccharide 309a and tetrasaccharide 307a 307a provided 85% and 73% of inhibition at the same concentration, respectively. Based on these provided 85% and 73% of inhibition at the same concentration, respectively. Based on these results, results, compound 314b was conjugated to CRM197 and employed for mice immunization. Sera analysis compound 314b was conjugated to CRM197 and employed for mice immunization. Sera analysis showed that glycoconjugate 314c was able to induce specific anti-Lam IgG Abs titers significantly showed that glycoconjugate 314c was able to induce specific anti-Lam IgG Abs titers significantly higher and homogeneous compared to Lam-CRM197 conjugate. This result suggested that the linear higher and homogeneous compared to Lam-CRM197 conjugate. This result suggested that the linear hexasaccharide β-(1-3) glucan 314b is long enough to cover the dominant epitope of C. albicans. hexasaccharide β-(1-3) glucan 314b is long enough to cover the dominant epitope of C. albicans. In 2015, Liao et al. [248] reported the synthesis of longer β-(1-3) glucan chains such as fragments In 2015, Liao et al. [248] reported the synthesis of longer β-(1-3) glucan chains such as fragments 316a–319a (Figure 34). Hexasaccharide 316a was assembled with a [4+2] glycosylation reaction from 316a–319a (Figure 34). Hexasaccharide 316a was assembled with a [4+2] glycosylation reaction donor 320 and compound 321 after cleavage of 2-Naphthylmethyl ether (Nap) group. Longer from donor 320 and compound 321 after cleavage of 2-Naphthylmethyl ether (Nap) group. Longer oligomers 317a, 318a and 319a were obtained via [4+4] (from 320), [8+2] (from 322 and 321) and [8+4] oligomers 317a, 318a and 319a were obtained via [4+4] (from 320), [8+2] (from 322 and 321) and (from 322 and 320) glycosylation strategies, respectively (Figure 34). Oligomers 316a–319a were [8+4] (from 322 and 320) glycosylation strategies, respectively (Figure 34). Oligomers 316a–319a were conjugated to KLH and KLH conjugates (316b–319b) were injected in mice, raising high IgG1 titers conjugated to KLH and KLH conjugates (316b–319b) were injected in mice, raising high IgG1 titers with significant statistical difference between hexasaccharide 316b and longer oligomers 317b–319b. with significant statistical difference between hexasaccharide 316b and longer oligomers 317b–319b. Octasaccharide 317b resulted the most immunogenic and protective compared to decasaccharide Octasaccharide 317b resulted the most immunogenic and protective compared to decasaccharide 318b and dodecasaccharide 319b. This result encouraged the authors to perform fungal challenge 318b and dodecasaccharide 319b. This result encouraged the authors to perform fungal challenge experiment in mice with glycoconjugate 317b using C. albicans fungus strain SC5314. In this experiment in mice with glycoconjugate 317b using C. albicans fungus strain SC5314. In this experiment, experiment, 11 mice were immunized with 317b and, after challenge, 4 mice (about 34%) were 11 mice were immunized with 317b and, after challenge, 4 mice (about 34%) were unaffected, unaffected, suggesting their protection from C. albicans. suggesting their protection C. albicans. More recently, Liao et from al. [249] reported similar immunological studies of fragments 323a–325a More recently, Liao et al. [249] reported similar immunological studies of fragments 323a–325a containing the dominant linear octasaccharide and β-(1-6) and β-(1-3) branches with different chain containing the dominant octasaccharide β-(1-6) β-(1-3) branches with chain length (Figure 35). KLHlinear conjugates 323b andand 324b wereand more immunogenic thandifferent 325b but the length (Figure 35). KLH conjugates 323b and 324b were more immunogenic than 325b but the majority majority of elicited Abs were against the common β-glucan motif. In addition, conjugates 323b and of elicited werelong-term against the common β-glucan motif.infection In addition, conjugates 323b and 325b conferAbs similar protection against C. albicans (survival rate about 37%, 30 325b days confer similar long-term protection against albicans infection ratethat about 37%, 30 days after after challenge for both conjugates). These C. data confirmed the(survival hypothesis linear β-(1-3) glucans challenge forwithout both conjugates). These the hypothesis that linear β-(1-3) glucans of of 6–8 units branches can coverdata the confirmed dominant epitope of C. albicans. 6–8 units without branches can cover the dominant epitope of C. albicans.
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Figure 34. Hexa-, octa-, deca- and dodecasaccharide of β-glucan reported in Reference [248]. Figure 34. Hexa-, octa-, deca- and dodecasaccharide of β-glucan reported in Reference [248]. Figure 34. Hexa-, octa-, deca- and dodecasaccharide of β-glucan reported in Reference [248]. Figure 34. Hexa-, octa-, deca- and dodecasaccharide of β-glucan reported in Reference [248].
Figure 35. Structure of fragments 285a–287a and their KLH conjugates [249]. Figure 35. Structure of fragments 285a–287a and their KLH conjugates [249].
Figure 35. Structure Structure of of fragments fragments 285a–287a 285a–287a and and their 35. their KLH KLH conjugates conjugates [249]. [249]. 14.2. AspergillusFigure fumigatus
14.2. Aspergillus Aspergillus fumigatus fumigatus fumigatus causes severe and usually fatal invasive aspergillosis infections in 14.2. Aspergillus 14.2. Aspergillus fumigatus immunosuppressed hospitalized patients. More than 90% of the cell wall of A. fumigatus comprises Aspergillus fumigatus fumigatus causes causes severe severe and usually usually fatal invasive invasive aspergillosis aspergillosis infections infections in in Aspergillus Aspergillus fumigatus causes severe and andis present usually infatal fatal invasivevariable aspergillosis in polysaccharides, among which α-(1,3)-glucan percentages from 20infections to 40%. This immunosuppressed hospitalized patients. More than 90% of the cell wall of A. fumigatus comprises immunosuppressed hospitalized hospitalized patients. patients. More More than than 90% 90% of the the cell cell wall of fumigatus comprises immunosuppressed wallstudy of A. A.of fumigatus comprises virulence factor suffers of poor solubility in water, makingofdifficult the its immunological polysaccharides, among among which which α-(1,3)-glucan α-(1,3)-glucan is is present present in in percentages percentages variable variable from from 20 20 to to 40%. 40%. This This polysaccharides, polysaccharides, among which α-(1,3)-glucan is present in percentages variable from 20 to This properties. The use of shorter synthetic fragments could overcome the solubility issue.40%. Indeed, virulence factor suffers of poor solubility in water, making difficult the study of its immunological virulence factor suffers of solubility in making the of its virulence of poor poorthe solubility in water, water, making difficult difficult the study study its immunological immunological Komarovafactor et al. suffers [250] reported synthesis of pentasaccharide fragment 326a of (Figure 36), the BSAproperties. The use of shorter synthetic synthetic fragments fragments could could overcome overcome the the solubility solubility issue. issue. Indeed, Indeed, properties. The use of shorter properties. The (326b) use ofand shorter syntheticasfragments could overcome the solubility Indeed, glycoconjugate its evaluation a vaccine candidate. Pentasaccharide 326aissue. was prepared Komarova et al.al.[250] reported the the synthesis of pentasaccharide fragment 326a (Figure 36), the BSAKomarova etal. [250] reported synthesis of pentasaccharide fragment 326a 36), (Figure 36), Komarova [250] reported the donor synthesis pentasaccharide 326a (Figure the BSAvia a [3+2] et glycosylation between 327ofand acceptor 328. fragment These protected fragments were in glycoconjugate (326b) and its evaluation as a vaccine candidate. Pentasaccharide 326a was prepared the BSA-glycoconjugate (326b) and its evaluation ascandidate. a vaccinePentasaccharide candidate. Pentasaccharide 326a glycoconjugate (326b) and its evaluation as a vaccine 326a was prepared turn synthesized from monomers 329 and 330. The use of benzoyl group at 6-OH as remote via aprepared [3+2] glycosylation between donor 327 and acceptor 328. These protected fragments were in was via a [3+2]between glycosylation between donor 327 acceptor 328.fragments These protected via a [3+2] glycosylation donor 327 and acceptor 328.and These were in partecipating group during glycosylation reactions facilitated theprotected formation of α-linkages. turn synthesized from monomers 329 and 330. The use of benzoyl group at 6-OH as remote fragments were infrom turn monomers synthesized329 from monomers and 330. The use of at turn synthesized 330.eliciting The 329 use of benzoyl at benzoyl 6-OH polyclonal asgroup remote Glycoconjugate 326b was immunized inand mice, highly specificgroup and protective partecipating group during glycosylation reactions facilitated the formation of α-linkages. 6-OH as remote partecipating group during glycosylation reactions facilitated the formation of partecipating group during glycosylation reactions facilitated the formation of α-linkages. antibodies. Glycoconjugate 326b was immunized in mice, eliciting highly specific and protective polyclonal α-linkages. Glycoconjugate 326b was immunized in mice, eliciting highly and polyclonal protective Glycoconjugate 326b was immunized in mice, eliciting highly specific andspecific protective antibodies. polyclonal antibodies.antibodies.
Figure 36. Retrosynthetic analysis of pentasaccharide 326a, as reported in [250]. Figure 36. Retrosynthetic analysis of pentasaccharide 326a, as reported in [250].
Figure 36. analysis of pentasaccharide 326a, as reported in [250]. Galactomannan is Retrosynthetic a specific polysaccharide produced by A. as fumigatus of a linear Figure 36. Retrosynthetic analysis of pentasaccharide 326a, reported composed in [250]. mannan core partially branched with β-(1,5)-galactofuranoside (Galf) units via β-(1,6) or β-(1,3) Galactomannan is a specific polysaccharide produced by A. fumigatus composed of a linear Galactomannan a specific polysaccharide produced by A.differs fumigatus composed of cultures, a linear linkages (Figure 37) is [251]. The length of β-(1,5)-Galf branches among different Galactomannan is a specific polysaccharide produced by A.(Galf) fumigatus linear mannan core partially branched with β-(1,5)-galactofuranoside unitscomposed via β-(1,6)ofora β-(1,3) mannan core partially branched with β-(1,5)-galactofuranoside (Galf) units via β-(1,6) or β-(1,3) making difficult the identification of a common repeating unit for galactomannans. Recently, Kudoh mannan partially branched with β-(1,5)-galactofuranoside ) units viadifferent β-(1,6) or β-(1,3) linkages core (Figure 37) [251]. The length of β-(1,5)-Galf branches (Galf differs among cultures, linkages (Figure 37) [251]. lengthofofsignificant β-(1,5)-Galf branches differs among cultures, et al. [252] demonstrated theThe presence structural differences in bothdifferent the O-linked and making difficult the identification of a common repeating unit for galactomannans. Recently, Kudoh making difficult the identification of a common repeating unit for galactomannans. Recently, Kudoh N-linked oligosaccharide of A. fumigatus galactomannans, depending on growth conditions in et al. [252] demonstrated the presence of significant structural differences in both the O-linked and et al. [252] demonstrated the presence of significant structural differences in both the O-linked and N-linked oligosaccharide of A. fumigatus galactomannans, depending on growth conditions in N-linked oligosaccharide of A. fumigatus galactomannans, depending on growth conditions in
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linkages (Figure 37) [251]. The length of β-(1,5)-Galf branches differs among different cultures, making difficult the identification of a common repeating unit for galactomannans. Recently, Kudoh et al. [252] Molecules 2018, 23, x FOR PEER REVIEW 37 of 52 demonstrated the presence of significant structural differences in both the O-linked and N-linked oligosaccharide A. fumigatus galactomannans, on growth conditions in different culture different cultureofmedia. Moreover, the authorsdepending revealed new structural elements of A. fumigatus media. Moreover, the authors revealed new structural elements of A. fumigatus galactomannan, the like galactomannan, the like the presence of β-(1,6)-linked Galf residues in addition to the β-(1,5)-linked the of(Figure β-(1,6)-linked Galf residues in addition to the β-(1,5)-linked Galf residues (Figure Molecules 2018, 23, x FOR PEER REVIEW 37 of 52 37). Galfpresence residues 37). different culture media. Moreover, the authors revealed new structural elements of A. fumigatus galactomannan, the like the presence of β-(1,6)-linked Galf residues in addition to the β-(1,5)-linked Galf residues (Figure 37). f
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Figure 37. 37. A. A. fumigatus fumigatus galactomannan structure, structure, underlining the presence presence of of β-(1,6)-linked β-(1,6)-linked Galf Galf f f Figure galactomannan underlining the f f residues in addition to the β-(1,5)-linked Galf residues, as reported in [252]. residues in addition to the β-(1,5)-linked Galf residues, as reported in [252]. Figure 37. A. fumigatus galactomannan structure, underlining the presence of β-(1,6)-linked Galf
The residues galactofuranosides (Galf) are not Galf presents inasmammalian cells and they are therefore used in addition to the β-(1,5)-linked residues, reported in [252]. The galactofuranosides (Galf ) are not presents in mammalian cells and they are therefore used for for diagnosis of human infections. In addition, they can be advantageously used as antigen diagnosis The of human infections. In addition, can be advantageously usedthey as antigen candidates (Galf) are notthey presents in mammalian cells and are therefore used for candidates forgalactofuranosides an anti A. fumigatus vaccine. an anti fumigatusofvaccine. forA.diagnosis human infections. In addition, they can be advantageously used as antigen Recently, the Nifantiev group reported the synthesis of pentasaccharide fragments of the candidates the for an anti A. fumigatus Recently, Nifantiev group vaccine. reported the synthesis of pentasaccharide fragments of the galactomannan containing the β-(1,5)-linked galactofuranoside chain attached to O-3 or O-6 of a Recently, the Nifantiev group reportedgalactofuranoside the synthesis of pentasaccharide fragments of O-6 the of a galactomannan containing the β-(1,5)-linked chain attached to O-3 or mannopyranoside residue (GM-1 331a, GM-2 galactofuranoside 332a and GM-3 333a) [253] andtofragments 334a–343a galactomannan containing the β-(1,5)-linked chain attached O-3 or O-6 of a mannopyranoside residue (GM-1 331a, GM-2 332a and GM-3 333a) [253] and fragments 334a–343a (Figure 38) [254]. In particular, pentasaccharide 331a was achieved via and [2+3]fragments glycosylation between mannopyranoside residue (GM-1 331a, GM-2 332a and GM-3 333a) [253] 334a–343a (Figure 38) [254]. In particular, pentasaccharide 331a was achieved via [2+3] glycosylation between disaccharide donor and trisaccharide acceptor 345was (Figure 38),via in [2+3] turn glycosylation prepared from donor 344 (Figure 38) [254].344 In particular, pentasaccharide 331a achieved between disaccharide donor 344 and trisaccharide acceptor 345 (Figure 38), in turn prepared from donor 344 and mannosyl acceptor 346. 344 was345 synthesized monosaccharides 347 and disaccharide donor 344 andDisaccharide trisaccharide acceptor (Figure 38),from in turn prepared from donor 344 348, and mannosyl acceptor 346. Disaccharide 344 was synthesizedfrom from monosaccharides 347 348, and 348, and mannosyl acceptor 346. Disaccharide 344 was synthesized monosaccharides 347 and both derived from allyl galactoside 350 through furanoside 349 (Figure 38). both derived fromfrom allylallyl galactoside 350 349(Figure (Figure38). 38). both derived galactoside 350through throughfuranoside furanoside 349 f
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Figure 38. Synthetic fragment of A. fumigatus galactomannans reported in [254,255].
Figure 38. Synthetic fragment of A. fumigatus galactomannans reported in [254,255]. Figure 38. Synthetic fragment of A. fumigatus galactomannans reported in [254,255]. The authors showed [255] that two mAbs (7B8 and 8G4), recognizing galactomannan in A. fumigatus, were elicited in mice after immunization with glycoconjugate 331b. Glycoarray analysis The authors showed [255] that two mAbs (7B8 and 8G4), recognizing galactomannan in A. performed with synthetic biotinylated oligosaccharides 331c–343c and mAbs 7B8 and 8G4 showed fumigatus, were elicited in mice after immunization with glycoconjugate 331b. Glycoarray analysis high affinity towards pentasaccharide 331c and heptasaccharide 343c while no recognition was
performed with synthetic biotinylated oligosaccharides 331c–343c and mAbs 7B8 and 8G4 showed high affinity towards pentasaccharide 331c and heptasaccharide 343c while no recognition was
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The authors showed [255] that two mAbs (7B8 and 8G4), recognizing galactomannan in A. fumigatus, were elicited in mice after immunization with glycoconjugate 331b. Glycoarray analysis performed with synthetic biotinylated oligosaccharides 331c–343c and mAbs 7B8 and 8G4 showed high affinity towards pentasaccharide 331c and heptasaccharide 343c while no recognition was observed for mono- and disaccharides 334c–338c. Finally, confocal microscopy was used to evaluate the binding between the two mAbs and A. fumigatus and a series of other fungi and bacteria. mAbs 7B8 and 8G4 were selective for A. fumigatus, suggesting that they can be used as reagents for immune diagnostics. Recent advances in galactofuranoside synthesis [205,232,233] should facilitate the access to galactomannan fragments, thus opening the way to anti A. fumigatus vaccines based on synthetic Galf oligomers. 15. Conclusions Pathogen surface glycans are promising vaccine targets due to their crucial role in adhesion to host tissues. Firstly, pathogen polysaccharides act as a protection barrier either by delaying the host’s immune response or by mimicking the host self-glycans. They are therefore essential for the survival of microorganisms in the blood and play a major role in their virulence. Secondly, most glycans are recognized by antigen presenting cells and triggers the innate immune response, starting the inflammatory process and eventually the antigen-specific adaptive immunity. Based on these considerations, the highly conserved polysaccharides (PS) exposed on pathogens cells surface are important virulence factors and have been used as antigens for vaccines development in a range of infectious diseases, including meningitis, pneumonia, otitis media, sepsis, infectious diarrheas, etc... The first-generation polysaccharide vaccines made of purified T cell-independent polysaccharide antigens have limited efficacy in infants, in the elderly and immunocompromised individuals and, in general, they don’t result in B cell-mediated immunological memory. The second-generation polysaccharide vaccines consist of native PS, produced and purified from natural sources and chemically conjugated to immunogenic carrier proteins able to elicit a T cell-dependent immune response (glycoconjugate vaccines). The purified native PS displays heterogeneity of chain length and may contain copurified endotoxin or other polysaccharides, introducing complications into the manufacturing process. Recent advances in carbohydrate chemistry has opened the way to a third-generation of polysaccharide vaccines, based on the use of synthetic oligosaccharides conjugated to carrier protein. The breakthrough in synthetic vaccines was carried out for H. influenzae type b in 2004, when the Vérez Bencomo research team in Cuba synthesized the first synthetic conjugate vaccine currently available under the trade name Quimi-Hib [9–62]. This approach is based on the hypothesis that protein conjugates of synthetic oligosaccharide fragments, smaller than the native PS, can elicit PS-specific and protective antibodies. The main challenge for the development of effective glycoconjugate vaccines is the determination of the carbohydrate antigen chain length and the sequence of the minimal protective immunogenic fragment of the antigen, a process often referred to as epitope mapping. The optimal carbohydrate antigen chain length needed for inclusion in a glycoconjugate vaccine, for example, cannot be predicted a priori for any given bacterial species. Despite the old paradigm established by Kabat [256], stating that immunogenic glycan epitopes usually comprise structures not longer than six-eight sugar units, the effect of the carbohydrate chain length on the immunogenicity of the corresponding protein conjugate is strictly case-dependent. There are many reported cases where both long chain glycans and oligosaccharides as short as tetrasaccharides have been shown to possess the minimal structural requirements for raising protective immunity, and several examples are illustrated throughout this review. In this regard, synthetic chemistry is a formidable tool to define the role of this crucial parameter, and more generally speaking of each structural variable affecting the immunogenicity and efficacy of vaccine candidates. Over the last years, much progress has been made. The rational design of carbohydrate antigens and the use of increasingly sophisticated synthetic strategies has made the preparation of even highly complex microbial glycans possible. In addition, the application of modern site-selective protein conjugation strategies [6,257,258] (a subject
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not addressed in the present review), has enabled the preparation of chemically defined glycoconjugate vaccine candidates featured by robust structure-immunogenicity relationship, which are expected to display improved safety and efficacy profiles [259]. These new achievements have certainly opened up new perspectives and hope for the prevention of severe bacterial and fungal infections still affecting the pediatric population worldwide. We truly wish that these promising results translate into new and efficient vaccines in the next few years. Author Contributions: C.C., O.P., and L.L. wrote the paper. Funding: We gratefully acknowledge funding from the H2020-MSCA-ITN-2015 “Glycovax” under Grant agreement No. 675671. Conflicts of Interest: The authors declare no conflict of interest.
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