Despite the enormous progress achieved by modern medi-cine, numerous diseases still have a profound impact on pub-lic health. Infectious diseases caused by a variety of microor-ganisms (viruses, fungi and parasites) and bacteria are aglobal major concern and, because of the emergence, for in-stance, of multidrug resistance, not only in developing coun-tries. The development of preventative therapies, such as therational design of novel and more efficient vaccines, mightoffer a solution to this state of affairs and other associateddrawbacks. Vaccination is considered by the World HealthOrganization to be the most cost-effective strategy for con-trolling infectious disease, because it should confer long-termprotective immunity in the population. A second consider-ation involves cancer. The outstanding progress achieved inthe identification and structural characterization of tumour-associated antigens has prompted their employment in tu-mour immunotherapy, on the basis of the observation thattumour cells possess specific antigens that can be recognizedby an immune system appropriately conditioned to the task.
Contents1. Introduction2. Immune response to carbohydrates3. Antibacterial vaccines3.1. Haemophilus influenzae3.2. The Shigella group3.3. Streptococcus pneumoniae3.4. Borrelia burgdorferi3.5. Neisseria meningitidis3.6. Vibrio cholerae3.7. Mycobacterium tuberculosis3.8. Bacillus anthracis4. Antiviral vaccines4.1. HIV4.1.1. Carbohydrate-based anti-HIV vaccines4.2. Influenza virus4.2.1. Carbohydrate-based anti-influenza vaccines5. Parasitic and helminth infections5.1. Infection by Plasmodium falciparum5.2. Infection by Leishmania spp.
[a] Dipartimento di Chimica Organica e Industriale, CISI andISTM-CNR, Università degli Studi di Milano,Via Venezian 21, 20133 Milano, Italy
Carbohydrates play key roles in many molecular recognitionphenomena and they can affect any kind of interaction withthe immune system. Saccharide-based antigens (bacterialcapsular polysaccharides or tumour-associated carbohydrateantigens, for instance) have therefore been studied and em-ployed in the formulation of vaccines. In recent years therehas been increasing use of synthetic saccharide antigens forthe formulation of vaccine candidates. These structures areindeed chemically well defined, devoid of biologic contami-nants and, in principle, available in large amounts, relativeto materials extracted from natural sources. In addition, syn-thetic saccharide antigens can also serve as haptens in pro-tein conjugates, eliciting highly specific antibodies in animalmodels and humans. The great potential of synthetic saccha-ride antigens is attested to by the spectacular success of theCuban vaccine against Haemophilus influenzae type b. Herewe review the major advances in the development of syn-thetic carbohydrate-based vaccines targeted against infec-tious diseases and cancer.
5.3. Infection by Tripanosoma cruzi5.4. Infection by Toxoplasma gondii5.5. Infection by helminths6. Fungal infections6.1. Infection by Candida spp.6.2. Infection by Criptococcus spp.7. Anticancer vaccines7.1. Brief description of TACAs7.2. Glycoconjugate cancer vaccines7.3. Fully synthetic carbohydrate-based cancer vaccines8. Conclusions
1. Introduction
Carbohydrates constitute one of the major classes of bio-molecules, together with proteins and nucleic acids. How-ever, despite the crucial roles they play in numerous bio-logical recognition processes (e.g., bacterial and viral infec-tion, cancer metastasis and inflammatory reactions),[1]
carbohydrates are the least exploited as potential thera-peutic agents. All cells, for example, are coated with thicklayers of complex carbohydrates known as the glycocalyx,
L. Morelli, L. Poletti, L. LayMICROREVIEWin which the glycan components are present in many dif-ferent glycoforms, such as glycoproteins, proteoglycans, gly-colipids and glycophosphatidylinositol-linked proteins. Thecell glycocalyx is therefore involved in cell differentiation,recognition, adhesion and many other important events, in-cluding pathological developments. The exposure of carbo-hydrates on cells’ surfaces enables them to interact with theimmune system, acting as cell antigenic determinants. Onthe other hand, the dense surface distributions of oftenunique glycan structures on diverse pathogens and on ma-lignant cells make carbohydrates attractive vaccine targets.The use of carbohydrates to induce immunity, however, is arelatively new strategy, although the seminal finding thatthe pneumococcal antigens targeted by the immune systemare polysaccharides dates back to 1923.[2] The advent ofchemotherapeutics and antibiotics dampened enthusiasmfor the development of carbohydrate vaccines, but the emer-gence of multidrug resistance phenomena and their con-stant increase roused renewed interest. In 1983 PneumoVax(Merck and Co.), the first polysaccharide vaccine, was com-mercially launched. This vaccine was made up of unconju-gated capsular polysaccharide isolated from 14 serotypes ofthe Streptococcus pneumoniae bacterium, whereas the cur-rent version includes 23 out of approximately 90 knownserotypes.[3] In healthy adults, this vaccine induces goodprotection against most of the infections caused by thesepathogens. Polysaccharide-based vaccines are poorlyimmunogenic, however, in infants, young children (under 2years of age), the elderly and immunocompromizedpatients, whereas in adults they induce only short-lastingantibody responses and fail to generate conventional B-cell-mediated immunological memory.[4] The consequent limitedclinical efficacy of polysaccharide-based vaccines is largelyattributed to the T-cell-independent immune responses,which are typically triggered by repetitive carbohydrate an-tigens.[5]
Luigi Lay (centre) received his Ph.D. in Organic Chemistry in 1994 at the University of Milan,under the guidance of Prof. Giovanni Russo, working on the synthesis of oligosaccharides relatedto heparin/heparan sulfate glycosaminoglycans. In 1995 he moved to the Institut für OrganischeChemie, University of Konstanz (Germany) as a postdoctoral fellow, where he worked on thesynthesis of glycoconjugates of the lacto and neolacto series under the supervision of Prof. R. R.Schmidt. In 1996 he moved back to Milan and joined the Italian National Research Council as aresearcher assistant. In 2000 he was employed as a researcher at the University of Milan (Depart-ment of Organic and Industrial Chemistry). Since 2010 he is professor of organic chemistry atthe same university. His research activity currently addresses the area of the synthesis of oligosac-charides glycoconjugates endowed with immunological activity and the synthesis of sugar ana-logues (C-glycosides and C-phosphonates) with potential biological activity.
Laura Poletti (left) was born in 1970 in Milan, Italy. She graduated in Chemistry at the Università degli Studi of Milan in 1995, under thesupervision of Prof. Franco Cozzi. In 1999 she received her Ph.D degree in Industrial Chemistry, from the same University, under thesupervision of Prof. Giovanni Russo. During her graduate studies she worked on the synthesis of heparin-like oligosaccharides starting fromstrategically protected building blocks. In 2000 she moved to the Ronzoni Institute of Chemistry and Biochemistry, Milan, for a postdoctoralfellowhip dealing with the chemical modification of glycosaminoglycan polymers. In 2001 she went back to the University of Milan, whereshe was hired as a researcher in 2005. Her current research interest is focused on the synthesis of glycoconjugate compounds and theiranalogues as potential anti bacterial vaccines.
Laura Morelli (right) was born in Genova in 1983. She received her M.Sc. degree in Chemistry in 2008 from the University of Milan, whereshe conducted research focused on the preliminary studies for the synthesis of a small library of bidentate cholera toxin ligands, under thesupervision of Prof. Anna Bernardi. She is currently completing her PhD program under the supervision of Prof. Luigi Lay at the Universityof Milan. Her research is focused on the synthesis of phosphodiester-linked oligomers of bacterial capsular polysaccharides.
Polysaccharide immunogenicity can be strongly en-hanced by conjugation to an immunogenic carrier protein,as suggested in 1931 by Avery and Goebel[6] and more re-cently successfully adopted to target some bacterial infec-tions associated with high mortality rates.[7] Glycoconjugateantigens are able to invoke T cell recruitment and immuneB cell memory specifically directed toward the carbo-hydrate.[8] Glycoconjugate vaccines exert useful immunolog-ical activities, even in persons in high-risk groups, and theirgeneration has been one of the greatest success stories inthe biomedical sciences.[9] Several conjugate versions ofpolysaccharide vaccines are now either commercially avail-able or in development.[10]
The carbohydrate-based antigens necessary for inclusionin a vaccine, however, are not readily available from naturalsources. In particular, the heterogeneity of naturally occur-ring glycans, obtained by means of challenging isolation,purification and identification techniques, and unavailabil-ity of sufficient amounts of material are the main limita-tions to further expansion in this field. Consequently, thedevelopment of future-generation vaccines is largely basedon recourse to fully synthetic carbohydrate antigens withdefined compositions, affording highly reproducible bio-logical properties. Synthetic carbohydrate vaccines have im-portant advantages over those isolated from natural sourcesbecause synthetic glycans can, in theory, be produced ashomogeneous compounds in a controlled manner with littleor no batch-to-batch variability. Synthesis, taking advan-tage of the glycosylation methods developed in the last dec-ades,[11] including automated synthesis,[12] allows the con-struction of complex oligosaccharide fragments. The finalproduct can be expected to have reliable structural integrityand appropriate purity for clinical application. Moreover,chemical modifications to the carbohydrate domain can beintroduced during the optimization of the vaccine con-struct, an option often unavailable when the antigen has to
Synthetic Oligosaccharide Antigens for Vaccine Formulation
be isolated from natural sources. Because of the danger ofcontaminating immunogens or disease-causing microbesthat can occur in vaccines derived from live cultures, fullysynthetic antigens also possess better safety profiles.
Although there are alternative methods for generatingglycoconjugate vaccines based on chemical manipulation ofisolated saccharide antigens or their fragments obtained bycontrolled hydrolytic depolymerization, this review focusesexclusively on vaccines or vaccine candidates containingcompletely synthetic carbohydrate antigens.
First and foremost, the next section briefly summarizesthe general features of the immune response to carbo-hydrates. The aim is to define some fundamental terms usedextensively throughout this review, in order to introducenon-specialized readers to the fascinating world of glycoim-munology.
In the subsequent sections we report on major advancesin the preparation of synthetic carbohydrate-based vaccinesdesigned to target bacterial, viral, fungal and parasitic in-fections, as well as cancer.
2. Immune Response to Carbohydrates
An antigen is any molecule perceived by the immune sys-tem as a foreign invader or simply as potentially dangerousfor the host. The immune system responds to antigens byeliciting a suitable immune response. More specifically, pro-
Figure 1. Diagramatic representation of the immune response.
tective immunity against pathogen exposure is achieved bythe integration of two distinct arms of the immune re-sponse: the innate and the adaptive (antigen-specific) re-sponses. The innate response is rapid and unspecific; it ismediated by antigen-presenting cells (APCs) and establishesthe first line of immune defence. It acts during the earlystages of infection (within minutes), detecting and re-sponding to pathogen-associated molecular patterns(PAMPs), which are structurally and chemically diversecompounds highly conserved in pathogens and absent intheir multicellular host. In contrast, the adaptive response,which is mediated by B and T lymphocytes, recognizespathogens with high affinity, providing the fine antigenicspecificity required for complete elimination of the infectiveagent and the generation of the immunological memory.
The establishment of adaptive immunity, however, typi-cally takes days to weeks to become effective. APCs (andin particular dendritic cells, DCs) provide a crucial bridgebetween the two responses. APC surfaces have plenty ofpattern-recognition receptors (PRRs), including the re-cently discovered Toll-like receptor family (TLRs), that canrecognize a huge variety of PAMPs. PRR stimulation cre-ates the necessary pro-inflammatory context (expression ofcostimulatory molecules and secretion of soluble cytokinesand chemokines), leading to full maturation of DCs, anti-gen uptake and intracellular processing (Figure 1, a). Ma-ture DCs migrate to the draining lymph nodes, where they
L. Morelli, L. Poletti, L. LayMICROREVIEWprime naive T cells, thus triggering and amplifying theadaptive arm of the immune response. The crucial featureof the T cell activation/differentiation process is the immu-nological synapse, initiated by formation of a ternary com-plex between MHC (major histocompatibility complex,class I or II, on DC surface), antigen and T cell receptor(TCR) on the T cell surface, followed by further specificinteractions. Depending on the antigen exposed on the DCsurface, the immunological synapse might induce the acti-vation of cytotoxic T lymphocytes (CTL, or CD8+ T cells,in cases in which the antigen is presented by a class IMHC), effector T cells that destroy target cells infected byintracellular viruses or bacteria, and/or the proliferation ofhelper T cells (Th, or CD4+ T cells, in cases in which theantigen is presented by a class II MHC). In turn, activatedTh cells elicit a conventional T-cell-dependent immune re-sponse by interacting with resting B cells through a class IIMHC and driving their proliferation and differentiationinto plasma cells (antibody-forming cells, mainly producinglow-affinity IgM-type antibodies) and memory B cells. Un-like plasma cells, memory B cells survive for a long time inthe body and respond rapidly to subsequent exposures ofantigen by secreting high-affinity IgG antibodies.
Although most antigens, especially proteins and deriva-tives, are T-dependent immunogens (i.e., they induce T cellactivation through MHC II-restricted pathways), immuneresponses to polysaccharides are typically T-cell-indepen-dent. Because of their polymeric structures, polysaccharidesbind several B cell receptors (BCRs) simultaneously, leadingto direct activation of B cells without cooperation fromT cells (Figure 1, b).[5] As a result, the hallmark immuneresponse to carbohydrates is an exclusively primary immuneresponse with low-affinity IgM production and no classswitch to high-affinity IgG antibodies. Moreover, pure poly-saccharides cause immune responses of relatively short du-ration, and they do not induce immunological memory (i.e.,they fail to invoke a booster effect). T-cell-independentpolysaccharides can be converted into T-cell-dependentimmunogens through covalent attachment to carrier pro-teins. The protein carrier incorporates T cell epitope pept-ides, which facilitate uptake and processing of the glycocon-jugate by APCs, enhancing the presentation of the carbo-hydrate antigen for activation of helper T cells. In this wayimmunological memory is established, raising a strong, du-rable and protective immune response from early child-hood. Typical examples of immunogenic carrier proteins in-clude bovine serum albumin (BSA) or its human variantHSA, keyhole limpet hemocyanin (KLH), bacillus Cal-mette–Guérin (BCG), CRM197 (a nontoxic variant of diph-theria toxin) and tetanus toxoid (TT).
Furthermore, in order to achieve optimal host protec-tion, a vaccine setting should include a component (adju-vant) capable of amplifying the immune response. In par-ticular, because saccharide antigens are often poorlyimmunogenic, carbohydrate-based vaccines need adjuvantsto improve their efficiencies and the quality and specificityof their immune responses. Typical immunoadjuvantswidely employed in vaccine settings are complete and in-
complete Freund’s adjuvants,[13] Detox, QS-21 (a saponinextracted from the bark of the Quillaja saponoria tree), andmonophosphoryl lipid A (MPLA). The administration ofthese strong immunoactive species can cause undesired sideeffects, however, and milder and safer lipopeptide-based im-munoadjuvants have been employed in many vaccine candi-dates. In particular, a number of derivatives of the lipopept-ide tripalmitoyl-S-glyceryl-cysteine (Pam3Cys, Figure 2)have been covalently linked to other components and in-cluded as “built-in” immunoadjuvants in vaccine con-structs.
Figure 2. Structure of Pam3Cys immunoadjuvant.
Adjuvants are perceived as “danger signals” after bind-ing to PRRs, and stimulate the activation and maturationprocess of APCs, thus enhancing the speed and durationof both the innate and the adaptive immune responses. Inparticular, adjuvants function as immune potentiators, pro-viding the pro-inflammatory context necessary for optimalantigen-specific immune activation and amplifying the in-nate immune response. After the discovery of the TLR fam-ily, it was shown that many PAMPs, as well as syntheticadjuvants, activate DCs upon stimulation of a specificTLR. These findings suggested that TLRs are essential inlinking innate and adaptive immunity throughout the entirecourse of the host defence response, because they are in-volved in multiple immunostimulatory activities. TLRs cantherefore be defined as general adjuvant receptors in thebody. On the other hand, adjuvants can also act as deliverysystems to localize vaccine components and to target themto APCs.
3. Antibacterial Vaccines
Among the human pathogens, there are a large numberof bacterial species that cause serious public health con-cerns. Despite the massive efforts directed towards eradicat-ing some infectious diseases, most still represent majorcauses of morbidity and mortality, as well as of the low lifeexpectancies at birth in the developing countries. Tradi-tional antibacterial vaccines have mainly consisted of liveattenuated pathogens, whole inactivated organisms or inac-tivated bacterial toxins. Although these agents have beensuccessful for vaccine development, leading to induction ofantibodies that neutralize viruses or bacterial toxins, inhibitthe binding of pathogens to cells or promote their uptakeby phagocytes, they can also give rise to undesirable sideeffects and safety problems. As a result of these limitations,several new approaches to vaccine development have
Synthetic Oligosaccharide Antigens for Vaccine Formulation
emerged. Some of these target pathogen-specific structuresand could offer significant advantages over more traditionalmethodologies.
The surfaces of bacterial pathogens are covered withdense arrays of oligo- and polysaccharides that, besidesconferring mechanical stability to the cell membranes of mi-croorganisms, are crucial protective antigens and virulencefactors. Encapsulated bacteria, for example, possess poly-saccharide coats (capsules) surrounding the bacterial cellsthat are essential for their pathogenicities, exerting protec-tive functions against the host’s immune defence. As a re-sult, infectious diseases from encapsulated bacteria are stillthe third leading cause of death in the world. A large bodyof literature data indicates that carbohydrate-specific anti-bodies are predominantly responsible for protection againstbacteria that feature either capsules or lipopolysaccharideson their surfaces, suggesting that vaccines consisting ofpurified pathogen-associated saccharide antigens might beeffective in conferring protection against infectious diseases.Oligosaccharide-based vaccines present advantages overconventional protein-based vaccines. Firstly, because the ex-pression of carbohydrates is not under direct genetic con-trol, cell surface oligosaccharides are highly conservedstructures. Furthermore, pathogenic cells’ surfaces typicallydisplay characteristic carbohydrate portions crucial for in-teractions with host structures. Nevertheless, some bacteriaexpress carbohydrate structures that have close similarityto mammalian tissue-specific structures and this molecularmimicry can induce tolerance by the host’s immune system.One general strategy to overcome this immunotolerance isto immunize with a chemically modified version of the gly-can, which is consequently perceived as a foreign antigenby the host. If the modification is sufficiently structurallyconservative, the elicited antibodies can cross-react with thenatural glycan expressed on the pathogen cell surface.
The main obstacle associated with carbohydrate-basedvaccines is that polysaccharides are T-cell-independent anti-gens and induce inadequate antibody responses (see Sec-tion 2). This notwithstanding, the first generation of carbo-hydrate-based vaccines, directed against encapsulated bacte-ria, has been developed through the use of free and purifiedcapsular polysaccharides. Important examples include thealready mentioned PneumoVax, the Vi-polysaccharide vac-cine against typhoid fever, and the four-component Neis-seria meningitidis groups A, C, Y and W135 vaccine. Thesevaccines were demonstrated to be highly effective in pre-venting disease in adults and older children, especially forshort-lasting exposure (travellers and soldiers in militarycampaigns). However, plain polysaccharides do not induceprotective immune responses in the immature immune sys-tems of newborns or children under two years of age. Theadvent of glycoconjugate vaccines, capable of conferringlong-term protective immunity even in high-risk groups,opened a new era in the field of vaccinology.[9]
The first licensed glycoconjugate vaccine was directedagainst Haemophilus influenzae type b (Hib) and its intro-duction in the routine vaccination programs of the devel-oped countries for children of pre-school age led to almost
complete disappearance of meningitis in newborns, infantsand children and, eventually, the whole population as a re-sult of “herd immunity”.[14] Other successful glycoconjugatevaccines – against Neisseria meningitidis type C,[15] for ex-ample – were developed, followed by the tetravalent menin-gococcal conjugate vaccine containing the A, C, Y andW135 serogroups of Neisseria meningitidis and currentlymarketed under the trade name Menactra. Between 800000and one million children under five years of age die annu-ally from Streptococcus pneumoniae infections.[16] Theglobal burden of disease and death caused by this bacte-rium led to the development of a heptavalent pneumococcalconjugate vaccine licensed under the trade name Prevnar in2000 and currently used in the USA in paediatric prac-tice.[17] Other pneumococcal conjugate vaccines includingmore virulent serotypes are currently under evaluation.[18]
Despite the extraordinary success of conjugate vaccinesconsisting of naturally derived polysaccharides (isolatedfrom the bacterial sources) or their fragments obtained bycontrolled acid hydrolysis and subsequent size fraction-ation, they have several shortcomings that could hampertheir acceptance by the Food and Drug Administration.The isolation and purification of the polysaccharide fromthe natural source is often a challenging task, leading to thepresence of biological contaminants and the possibility ofresidual toxicity with lipopolysaccharide-derived (LPS-de-rived) polysaccharides, thus raising severe problems of qual-ity assurance. Moreover, the conjugation chemistry is diffi-cult and can result in conjugates with ambiguities in com-position and structure. The recognition that fragments ofnative polysaccharides might act as haptens[19] in the formof protein conjugates by eliciting bacterial polysaccharide-specific antibodies stimulated recourse to fully synthetic oli-gosaccharide antigens in order to provide homogeneousand well-characterized conjugates in a more economicaland reproducible manner, as well as free from bacterial con-taminants. Reducing saccharide antigens are typically con-jugated by reductive amination, but this can destroy criticalepitopes, leading to a decrease in or loss of immunogenicity,especially in the case of small-sized oligosaccharides. Thestructural and immunological integrity of carbohydrate an-tigens can be preserved by incorporation into synthetic oli-gosaccharides of a readily activatable linker, in order to al-low selective conjugation to protein without interferencewith the antigenic epitope. Additional advantages of thesynthetic methods are: a) the opportunity to perform de-tailed SAR studies, and b) the potential for chemical modi-fications of the saccharide antigen, in order to evade immu-notolerance. The development of antibacterial glycoconjug-ate vaccines based on fully synthetic saccharide antigenshas been made possible by the enormous progress achievedin the chemical synthesis of oligosaccharides during lasttwo decades, in terms of protecting group strategies, newglycosylation methods and new protocols for protein andpeptide conjugation.[11a,12a,20]
The synthesis of homogeneous oligosaccharides withwell-defined structures and the evaluation of their immuno-genicities, as well as the chemical syntheses of bacterial oli-
L. Morelli, L. Poletti, L. LayMICROREVIEWgosaccharides published until the early part of 2002, havebeen reviewed by Pozsgay.[21] The next section of this reviewfocuses mainly on the synthesis and immunological evalu-ation of bacterial oligosaccharides from 2002 on.
3.1. Haemophilus influenzae
The first, and so far only, commercial vaccine containinga synthetic carbohydrate antigen was developed in Cubaagainst Hib [trade name Quimi-Hib (7, Scheme 1)].[22] Hibis a pathogen that, before the introduction of Hib polysac-charide conjugate vaccines in 1988, was the leading causeof bacterial meningitis in children in the USA. The intro-duction of the vaccine in developing countries, however, hasbeen slow because of its high cost and reduced availability.According to the WHO, Hib is currently responsible forapproximately three million serious illnesses and an esti-mated 386000 deaths per year in the developing world, mostin children under the age of five years.[23] Verez-Bencomoand Roy synthesized the β-d-ribose-(1,1)-d-ribitol-5-phos-phate H-phosphonate derivative 1 (Scheme 1), the repeatingunit of the Hib capsular polysaccharide, and the corre-sponding phosphodiester spacer-linked compound 2. Bymeans of a one-step polycondensation reaction with the useof H-phosphonate chemistry,[24] compounds 1 and 2 werecopolymerized in a 10:1 molar ratio to produce the oligo-mers 3, containing on average eight repeating units of ribos-ylribitol, in 80% yield after purification by size-exclusionchromatography. After hydrogenolysis, the spacer at the re-ducing end of the oligomers 4 was activated by treatmentwith N-hydroxysuccinimidyl 3-maleimidopropionate to pro-vide 5. The overall process could be carried out at a 100 gscale per batch. The vaccine prototype 6 was subsequentlyproduced by conjugation of 5 to thiolated HSA. This first
glycoconjugate was evaluated in an ELISA assay and wasfound to be strongly antigenic, being recognized by specificanti-Hib sera. The antigen 5 was next conjugated to themuch more immunogenic carrier protein tetanus toxoid(TT), which is also more suitable for use in humans. Conju-gation was achieved by exploiting the thiolation of TT ly-sine ε-amino groups, and the immunogenicity of the re-sulting glycoconjugate 7 was studied first in rabbits andthen in clinical trials in adults, children and infants. Afterfourteen years of experimentation, the final result has beena 99.7 % success rate in children, leading to Quimi-Hib be-coming part of Cuba’s national vaccination programme in2004.
The synthesis of capsular polysaccharide (CPS) frag-ments belonging to different serogroups of Haemophilus in-fluenzae, namely types a, c, d, e and f, as well as structuresfrom the LPS of the bacterium, have been the subject ofinvestigation by other groups.[21b,25] In particular, the cap-sule of Haemophilus influenzae type a (Hia), the cause of upto 10% of Haemophilus infections, is composed of a poly-mer of ribitol phosphate. Pozsgay recently reported the firstsynthesis of ribitol phosphate oligomers containing up totwelve repeating units.[26] The octamer and the dodecamerwere coupled to aminooxypropylated BSA, furnishing con-jugates with an average loading of eighteen copies of thehapten per protein molecule. However, no studies on theantigenicities and immunogenicities of these structures haveever been reported.
3.2. The Shigella Group
Shigellosis (bacillary dysentery) is endemic throughoutthe planet, although essentially a major health concern indeveloping countries, particularly in the paediatric popula-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
tion between one and five years old. Shigella consists offour distinct groups of non-encapsulated Gram-negativebacteria: S. dysenteriae, S. flexneri, S. boydii and S. sonnei.Each Shigella group includes several serotypes, except forS. sonnei, which has a single serotype.[27] As a result of itsexpression of the Shiga toxin (a potent cytotoxin that notonly worsens the intestinal symptomatology, but also causessystemic complications), S. dysenteriae type 1 is a majorcausative organism of diarrhoea and dysentery, as well asof epidemic disease in the world’s least developed countries.Nevertheless, there is still no licensed vaccine against thisbacterium, which is resistant to antibiotics in several coun-tries.[28] S. dysenteriae 1 expresses a lipopolysaccharide thatis a virulence factor and a protective agent. The O-specificpolysaccharide (O-SP) portion of its LPS is made up oftetrasaccharide repeating units (about 25 copies). Pozsgay’sgroup reported the first synthesis of the shifted tetrasaccha-ride 8[29] [α-l-Rha-(1�2)-α-d-Gal-(1�3)-α-d-GlcNAc-(1�3)-α-l-Rha, Figure 3] and its longer oligomers up tofour repeating units (the hexadecasaccharide 11).[30]
Figure 3. Structures of synthetic oligomers from S. dysenteriae 1 O-specific polysaccharide.
These compounds were conjugated to HSA through anew heterobifunctional spacer[30b] to provide neoglycopro-teins of diverse saccharide loading for use in immunologicalstudies on mice in comparison with a HSA conjugate com-posed of the O-SP prepared by acid hydrolysis of the LPSof S. dysenteriae 1.[31] The results showed that all the syn-thetic conjugates except for the tetrasaccharide elicited anti-LPS IgG antibodies at titres higher than those induced byadministration of the O–SP–HSA conjugate.[32] In particu-lar, the hexadecasaccharide 11 conjugate, with an averageof nine chains of saccharide per protein molecule, was themost immunogenic, although the dodecasaccharide 10 withthe same saccharide/protein ratio showed only a small de-crease in immunogenicity. More recently, the same authorssynthesized a panel of O-SP oligosaccharide conjugatesranging from hexa- to tridecasaccharide, differing in the na-ture of the monosaccharide at the nonreducing end of thesaccharide chain, to establish whether or not the identity ofthis terminal residue has an effect on anti-polysaccharideimmunogenicity.[33] The immunogenicities of these glyco-conjugates were found to be lower than that of the relatedO-SP conjugate, with the highest anti-LPS antibody levelsbeing elicited by conjugates with GlcNAc (decasaccharide)or Gal (hepta- and undecasaccharide) residues at their non-reducing termini.[34]
Shigella flexneri 2a is another major cause of the endemicform of shigellosis in developing countries. The O-SP moi-ety of S. flexneri 2a surface LPS is an essential virulencefactor and consists of a branched pentasaccharide repeatingunit. Mulard and co-workers reported the synthesis of anumber of S. flexneri 2a O-SP-related saccharide frag-ments.[35] In particular, they described the preparation ofthe thioacetylated penta-, deca- and pentadecasaccharides12–14 (Figure 4), corresponding to the monomer, dimerand trimer of the repeating unit, respectively.[36] Of the dif-ferent strategies that, in principle, could lead to the targetcompounds, the authors found the linear strategy to bemuch more advantageous, in terms of yields and number ofsteps, than various convergent routes. The antigenicities ofall the synthetic fragments were evaluated by ELISA assayin order to identify the protective immunogenic determi-nants specific to serotype 2a. The oligosaccharides 12–14were then covalently linked to maleimido-activated TT, re-sulting in conjugates with 12 saccharide chains on averageper protein molecule.[37]
Figure 4. Structures of synthetic oligomers from S. flexneri 2a O-specific polysaccharide.
The immunogenicities of the glycoconjugates were testedin mice and the pentadecasaccharide conjugate 14 elicitedthe highest immune response.[37] More recently, the sameauthors showed that the administration of pentadecasac-charide-induced anti-O-SP 2a antibodies protects micefrom Shigella infection, strongly suggesting that the trimerof the O-SP repeating unit is a functional mimic of thenative polysaccharide and might represent a potential can-didate for further development of a synthetic glycoconjug-ate vaccine against S. flexneri 2a.[38] In a further extensionof their work, the authors reported the preparation of fullysynthetic vaccine candidates in which the O-SP-related oli-gosaccharides 12 and 14 (B cell epitopes) are covalentlylinked to the influenza hemagglutinin peptide HA307–319 asa helper T cell epitope.[39] The B cell and the T cell epitopeswere covalently conjugated to the maleimide-functionalizedlipopeptide Pam3CysAlaGly (a TLR2 ligand known for itspowerful adjuvant properties) anchored on the surfaces ofpreformed liposomes. Immunization studies showed thatonly glycoliposomes displaying the pentadecasaccharidehapten induce a specific and long-lasting anti-S. flexneri 2a
L. Morelli, L. Poletti, L. LayMICROREVIEWimmune response, in accordance with previous observationssuggesting that a minimum of two repeating units of syn-thetic serotype 2a oligosaccharide is required for optimalmimicry of the O-SP epitopes.[40]
Oligosaccharide fragments of other S. flexneri serotypes,including pentasaccharides related to serotype 1a[41] andvarious fragments of serotype 3a[42] and serotype X O-SP,[41–43] have been synthesized. However, neither the evalu-ation of their antigenicities nor their conjugation to proteincarriers for immunogenicity studies have yet been reported.
3.3. Streptococcus pneumoniae
As mentioned above, infections from Streptococcus pneu-moniae are still a global health concern, especially in paedi-atric populations. After the licensing of Prevnar, varioussynthetic approaches were investigated, resulting in promis-ing vaccine candidates. Synthetic fragments from CPS ofS. pneumoniae type 3 and type 14, for instance, were conju-gated to a nontoxic variant of diphteria toxin (CRM197)and tested in mice.[44]
In a more recent study, sixteen different synthetic oligo-saccharide fragments of the S. pneumoniae 14 CPS, rangingfrom tri- to dodecasaccharides, were conjugated to CRM197
through squarate linkers and injected into mice to identifythe smallest immunogenic structure.[45] The results showedthat the branched Glc-(Gal)-GlcNAc trisaccharide elementof the tetrasaccharide 15 (Figure 5) is essential for induc-tion of polysaccharide-specific antibodies and that theneighbouring galactose unit at the nonreducing end clearlycontributes to the immunogenicity of the epitope. This in-vestigation confirmed that the branched tetrasaccharide 15could be a serious candidate for a synthetic oligosaccharideconjugate vaccine against infections caused by S. pneumon-iae 14.
Figure 5. Structure of the synthetic tetrasaccharide from CPS of S.pneumoniae type 14.
Demchenko and co-workers reported the chemical syn-thesis of oligosaccharides of the S. pneumoniae 6A and 6BCPS together with structural variants,[46] as well as the firstsynthesis of the repeating unit of the newly discovered sero-type 6C (Figure 6).[47] All of the synthetic oligosaccharideswere conjugated to BSA by the squarate method and theirantigenicities were tested with a rabbit antiserum used forpneumococcal serotyping.[47] The results showed that syn-thetic carbohydrate conjugates express epitopes found innative CPS of serotypes 6A, 6B and 6C and elicited broadly
cross-reactive antibodies that could be protective againstpneumococcal infection caused by the three serotypes ofS. pneumoniae 6.
Figure 6. Structures of repeating units from CPS of S. pneumoniaeserotypes 6A, 6B and 6C.
The CPS repeating unit of S. pneumoniae 19F, one of themost virulent pneumococcal serotypes, consists of the tri-saccharide 1-O-phosphate 16 shown in Figure 7. We[48] andothers[49] synthesized the saccharide portion of 16.
Figure 7. Structures of the repeating unit from CPS of S. pneumon-iae 19F and its carba-rhamno analogue.
In addition, the synthesis of 16 and of various phosphor-ylated fragments and oligomers was also reported.[50] Morerecently, we reported the synthesis of a carba analogue of16, in which a carba-rhamnose residue has been inserted inplace of the naturally occurring l-rhamnose unit (com-pound 17, Figure 7), in order to enhance the chemical sta-bility of the labile acetalic phosphodiester bridges.[51] Com-petitive ELISA assays demonstrated that both the carba an-alogue 17 and the corresponding natural trisaccharide 16are recognized by anti-19F antibodies, suggesting that thereplacement of the pyranose oxygen of l-rhamnose with amethylene group does not significantly affect the biologicalproperties.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
3.4. Borrelia burgdorferi
Lyme disease (LD) is a multisystemic chronic inflamma-tory disorder affecting the skin, eyes, joints, internal organsand nervous system. The etiologic agent of LD is the Gram-negative bacterium Borrelia burgdorferi, which is trans-mitted by ticks. B. burgdorferi is known to exhibit an ex-traordinarily high lipid content, and cholesteryl 6-O-palmi-toyl-β-d-galactopyranoside (18, Figure 8) was recently iden-tified as a major surface component.
Figure 8. Structure of the glycolipid major surface component ofB. burgdorferi.
Pozsgay and co-workers reported the syntheses of 18[52]
and of its bioconjugatable form 19,[53] which was covalentlylinked to aminooxypropylated bovine serum albumin(BSA). Very recently, a new synthesis of 18 together with alibrary of closely related glycolipids with variations in thefatty acid chain length and the cholesterol moiety was re-ported.[54] The antigenicities of the synthetic glycolipidswere investigated in immunoblots to determine the epitoperecognized by human antibodies, showing that galactose,cholesterol, and a fatty acid with a minimal chain lengthof four carbon atoms comprise the essential structure forrecognition.
3.5. Neisseria meningitidis
The Gram-negative encapsulated organism Neisseriameningitidis is the leading cause of bacterial meningitis.This invasive infection affects mostly infants, children andadolescents who do not possess specific antibodies. On thebasis of the chemical composition of the polysaccharidecapsule, 13 capsular serogroups of N. meningitidis have sofar been defined, but about 90% of infections are causedby the serotypes A, B, C, Y and W135. Although there aretwo licensed vaccines (bivalent A/C and tetravalent A/C/Y/W135) that are effective for persons older than two years,the formulation of suitable glycoconjugate vaccines againstN. meningitidis is required in order to improve immune re-sponses in young children. Accordingly, three monovalentgroup C conjugate vaccines and a tetravalent meningococ-cal conjugate vaccine against groups A, C, Y and W135 arecurrently on the market.
Although all N. meningitidis serotypes can cause epidem-ics, group A strains (MenA) are the main agents responsiblefor epidemics in sub-Saharan Africa (the so-called “menin-gitis belt”), where the annual disease incidence ranges from
1 to 8 ‰ of the population.[4a,55] The groups of Pozsgay[56]
and Oscarson[57] reported the syntheses of fragments (up tothe trimer) of the MenA CPS, consisting of (1�6)-linked2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate resi-dues (Figure 9).
Figure 9. Neisseria meningitidis A capsular polysaccharide repeat-ing unit (R = H or Ac).
In particular, Pozsgay also described the conjugation ofthe synthetic fragments to HSA, showing that a polyclonalanti-N. meningitidis A antiserum can recognize a monosac-charide fragment of its CPS. However, the MenA CPS suf-fers from poor stability in water, due to the inherent labilityof the anomeric phosphodiester groups bridging two N-acetyl mannosamine units. This structural property makesthe development of a fully synthetic glycoconjugate vaccinea challenging task. Access to synthetic analogues endowedboth with the immunological properties of the natural com-pounds (i.e., the ability to induce the production of anti-bodies that will cross-react with the bacterial capsule) andwith increased stability in water is therefore highly desirable.To this end, syntheses of phosphonoester-bridged frag-ments of the MenA CPS in which 1-C-phosphonates havebeen used as isosteric and nonhydrolysable analogues ofglycosyl 1-O-phosphates have been reported by Oscarson[58]
and by our group.[59] We also investigated the relative affin-ities of the synthetic molecules (the monomer 20, the dimer21 and the trimer 22, Figure 10) by means of a competitiveELISA assay, showing that the synthetic fragments contain-ing the unnatural interglycosidic phosphonoester linkageare recognized by a human polyclonal anti-MenA se-rum.[59b]
Figure 10. Phosphonoester-linked oligomers of Neisseria meningit-idis A capsular polysaccharide.
After these encouraging results, the synthetic oligomerswere conjugated to passivating thiols and employed for thefabrication of multivalent gold glyconanoparticles mimick-ing the bacterial capsule.[60] Interestingly, gold glyconano-particles displaying the synthetic Men A CPS fragmentsbind to specific anti-MenA antibody at least two orders ofmagnitude more strongly than the corresponding nonconju-
L. Morelli, L. Poletti, L. LayMICROREVIEWgated, monovalent oligomers.[61] Investigations to establishwhether these gold glyconanoparticles are also able to in-duce immune cell responses are in progress.
3.6. Vibrio cholerae
Cholera is a very serious enteric disease caused by theGram-negative bacterium Vibrio cholerae, marked by severediarrhoea that can lead to dehydration, hypotensive shockand death. More than 200 serogroups of V. cholerae, dif-fering in the structures of their O-specific polysaccharideportions of LPS, have been identified. Until 1992, when anew serogroup O:139 emerged, the O:1 strain was thoughtto be the only V. cholerae bacterium pathogenic in humans.V. cholerae O:1 occurs as two serotypes, termed Ogawa andInaba, the O-PSs of which are linear homopolymers of α-(1�2)-linked 3-deoxy-l-glycero-tetronamido-d-perosamine,the only structural difference being the 2-O-methylation ofthe upstream perosamine (4-amino-4,6-dideoxy-d-mann-ose) unit in the Ogawa serotype (Figure 11).
The Ogawa serotype, however, has been identified as thedominant determinant epitope and employed as target sac-charide antigen in the design of anticholera O:1 vaccine.The Kovác laboratory provided a major contribution to thisfield with the syntheses of a number of saccharide frag-ments (from mono- to hexasaccharides) of the O-antigen ofV. cholerae O:1, serotype Ogawa, and their protein conju-gates.[62] Immunological studies showed that none of the
Scheme 2. Synthesis of V. cholerae O:1 Ogawa hexasaccharide.
Figure 11. Structures of O-specific LPS portions of Vibrio choleraeO:1 Inaba and Ogawa serotypes.
neoglycoconjugates except for the hexasaccharide providedvibriocidal humoral responses, suggesting that the shorteroligosaccharides lack a conformational epitope provided bythe hexasaccharide. Moreover, the antiserum obtained frommice immunized with the hexasaccharide conjugate con-taining the lowest saccharide loading exhibited the highestprotective capacity.[63] In a subsequent study, the hexasac-charides corresponding to Ogawa and Inaba serotypes ofV. cholerae O:1 were prepared by a [4 + 2] assembling strat-egy, converted into their squaric acid derivatives and conju-gated with recombinant exotoxin A from Pseudomonasaeruginosa (rEPA).[64] The results of the immunological
Synthetic Oligosaccharide Antigens for Vaccine Formulation
tests highlighted the fact that Ogawa and Inaba LPS con-tain different immunodominant epitopes: Ogawa neoglyco-conjugates can boost Inaba-primed mice, but Inaba neogly-coconjugates cannot boost Ogawa-primed mice.[65] More re-cently, an improved synthetic approach to the Ogawa ter-minal hexasaccharide was described by the same authors.In this new strategy, the hexasaccharide 23 was assembledby a blockwise approach, as illustrated in Scheme 2.[66]
Firstly, the diazido disaccharide 24[67] was subjected toreduction with H2S, and the resulting 4-amino groups wereamidated with 2,4-di-O-acetyl-3-deoxy-l-glycero-tetronicacid to provide (after 2-O-levulinoylation of the nonreduc-ing perosamine unit) the ethyl thioglycoside disaccharidedonor 25. Separately, the disaccharide acceptor 28 was ob-tained by coupling of the donor 26 with the alcohol 27.Glycosylation of 28 with 25 gave the α-tetrasaccharide 29together with its β anomer in a ratio of 2:1. Much bettercontrol of the stereochemical course was achieved, however,when the glycosylation reaction was carried out underthermodynamic conditions (in a toluene/CH2Cl2 mixture atreflux), leading to the preferential formation of the thermo-dynamically more stable α-product (α/β = 5:1). Finally, thetetrasaccharide 29 was converted into the acceptor 30 andglycosylated with the disaccharide donor 31 (also obtainedfrom 24 in three steps) to provide the hexasaccharide 23after global deprotection. Once again, the α/β ratio wasclearly improved, from 3:1 to 5:1, by carrying out the reac-tion under thermodynamic control.
In 1992, a new serogroup of V. cholerae, termed O:139,caused a cholera epidemic in the Indian subcontinent andemerged as an additional threat to public health in de-veloping countries. V. cholerae O:139 expresses a CPS inwhich the repeating unit is identical to the O-PS portionof LPS, and both are virulence factors. The O-antigen ofV. cholerae O:139 is a hexasaccharide (Figure 12) consistingof two units of the rare deoxysugar 3,6-dideoxy-l-xylo-hexose (colitose, residues E and F) and a 4,6-cyclic phos-phate on the galactose moiety D. The Kovác group, usinga stepwise strategy, reported the synthesis of the disaccha-ride 32 (corresponding to the DC fragment), the trisaccha-ride 33 (corresponding to the FDC fragment) and tetrasac-charide 34 (corresponding to the FD(E)C fragment) of the
Figure 12. Hexasaccharide repeating unit of the O-antigen of Vib-rio cholerae O:139.
V. cholerae O:139 O-SP repeating unit (Figure 12) bearingan amino-functionalized spacer to allow conjugation tosuitable protein carriers.[68]
An alternative synthesis of the same V. cholerae O:139 O-antigen tetrasaccharide fragment FD(E)C, also in conjugat-able form, was developed by Oscarson and co-workers.[69]
Whereas the Kovác strategy allowed the preparation of anumber of smaller fragments, the main advantage of theOscarson approach is that the protected tetrasaccharide isgenerated as an ethyl thioglycoside and is therefore amena-ble to use as a glycosyl donor for the complete constructionof the full hexasaccharide.
3.7. Mycobacterium tuberculosis
Mycobacterium tuberculosis is the causative agent of tu-berculosis, which remains the leading cause of death frominfectious bacteria, despite the development of new treat-ments, and is second only to HIV as the leading cause ofadult death worldwide. Annually, tuberculosis causes al-most eight million new cases and two million deaths.[70] Inparticular, 90% of estimated deaths and 95% of new casesof tuberculosis each year occur in developing countries,which make up 85 % of the world’s population.[71] Theglobal health concern of tuberculosis is further worsenedby the emergence of multi-drug resistant strains, making theavailability of an improved and efficient anti-tuberculosisvaccine desperately needed. Unlike other bacterial patho-gens, M. tuberculosis is not characterized by a unique cap-sular polysaccharide that could be used as a vaccine target.Instead, the mycobacterial cell wall is rich in lipids andcomplex polysaccharides. The major component of the cellwall is a macromolecule of peptidoglycan covalently linkedthrough a phosphodiester group to an arabinan-capped lin-ear galactan. The arabinan cap is modified with variouslong-chain fatty acids, of 70–90 carbons in length, termedmycolic acids. Aside from the mycolyl–arabinogalactan–peptidoglycan complex, the cell wall also contains a varietyof noncovalently associated glycolipids. Among the mostabundant of these are a family of related glycophospholip-ids containing mannose, termed the phosphatidylinositolmannosides (PIMs), lipomannan (LM) and lipoarabinom-annan (LAM). Because of their biological importance, sig-nificant efforts have been devoted towards the synthesisM. tuberculosis capsular oligosaccharides.[72] Quite surpris-ingly, there has been no report yet on their use as possiblevaccine components, despite the finding that oligosaccha-rides derived from mycobacterial LAM and covalently con-jugated to TT and CRM197 proteins have proven to behighly immunogenic in animal models.[73] Important contri-butions of synthetic organic chemistry to the elucidation ofthe pathways leading to the biosynthesis of the mycobacte-rial cell wall include the first total syntheses of PIM2 andPIM6 by Seeberger’s group.[74] Fraser-Reid and co-workerssynthesized a LM component of mycobacterial LAM(Scheme 3).[75]
L. Morelli, L. Poletti, L. LayMICROREVIEW
Scheme 3. Retrosynthesis of the dodecasaccharidyl lipomannancomponent of mycobacterial LAM synthesized by Fraser-Reid.
The synthetic strategy is based on n-pentenyl orthoesters,easily prepared from d-mannose. These are employed bothas donors and as acceptors. The assembly of the branchedmannan chain is achieved by activation of the orthoestersthrough the use of the ytterbium triflate/N-iodosuccin-immide reagent system as a promoter. Seeberger’s groupsynthesized a dodecasaccharide fragment, corresponding tothe key saccharide portion of LAM, containing six α-Arafand six α-Manp residues in which the a key step is the [6 +6] coupling of the mannan and arabinan domains.[76] Aneven larger fragment (28 residues) made up of the inositol,15 α-Manp and 12 α-Araf residues was synthesized by aroute in which the key glycosylation was a [12 + 6] coupling
Figure 13. Structures of synthetic M. tuberculosis PIM1–PIM6.
between an arabinomannan donor and a mannosylated in-ositol acceptor.[77] Lowary’s laboratory reported the synthe-sis of a docosanasaccharide containing 22 arabinofuranoseresidues and corresponding to the repeating unit of the arab-inan domain of mycobacterial arabinogalactan.[78] Morerecently, Seeberger’s group reported the synthesis of all ofthe PIMs from PIM1 to PIM6 (Figure 13).[79] The synthesisof PIM3, outlined as an illustrative example in Scheme 4,utilizes the bicyclic and tricyclic orthoesters 38 and 37,respectively, and the mannosyl phosphates 40 and 39 as gly-cosylating agents. The mannosyl phosphate donor 40 wassynthesized from the bicyclic orthoester 38, which was inturn obtained from d-mannose. d-Mannose was likewisethe starting molecule for the preparation of the tricyclic or-thoester 37, which provided the mannosyl phosphate donor39. The myo-inositol acceptor 41 was obtained as previouslyreported by the same group.[74] The glycosylation of 41 with39, followed by coupling with the donor 40, afforded thepseudotrisaccharide acceptor 42. The PIM3 backbone wasfinally assembled by glycosylation of 42 with the donor 40to give the pseudotetrasaccharide 43. The phosphodiestermoiety was installed by deallylation of the myo-inositol resi-due followed by coupling with the H-phosphonate 44 andoxidation with I2 in pyridine/water. Global deprotection un-der Birch conditions afforded PIM3.
Each synthetic PIM was provided with a thiol linker forimmobilization on surfaces and carrier proteins for bio-logical and immunological studies. In particular, the syn-thetic PIMs were immobilized on microarray slides forstudy of their interactions with the dendritic-cell-specific in-tercellular adhesion molecule-grabbing nonintegrin (DC-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Scheme 4. Synthesis of the M. tuberculosis PIM3 component. NAP = 2-naphthylmethyl.
SIGN) receptor. Carbohydrate microarray is one of themost powerful high-throughput glycan-binding assays, en-abling the evaluation of low-affinity saccharide–protein in-teractions by exploitation of the multivalency effectachieved by displaying multiple glycans in an arrayed-chip-based format. A number of carbohydrate microarrays basedon covalent or noncovalent attachment protocols have beendeveloped.[80] These carbohydrate microarrays are formida-ble tools in vaccinology for identification of relevant glycantargets and evaluation of candidate immunogens.
DC-SIGN is an important PRR on dendritic cells thatcontributes to the initiation of the innate immune responseby antigen uptake, processing and later presentation on thesurface together with costimulatory molecules. Importantly,mycobacteria also use DC-SIGN as a receptor to enter den-dritic cells.[81] The synthetic oligosaccharides PIM5 andPIM6 showed the highest binding affinities to DC-SIGN.Moreover, the synthetic PIMs exhibited strong immunosti-mulatory activities during immunization experiments inmice. PIM6 was covalently linked to the model antigenKLH and the resulting conjugate induced a marked in-crease in anti-KLH antibodies in relation to KLH alone.The adjuvant properties of PIM6 were also confirmed bymeasurement of cytokine production.
All of these complex synthetic molecules are expected tomake important contributions in the search for carbo-hydrate antigens to be used for vaccination against M. tu-beculosis infections. Recently, a new mycobacterial antigenable to stimulate populations of CD1b-restricted humanT cells during infection with M. tuberculosis was charac-terized and identified as a diacylated sulfoglycolipid:acyl2SGL (45, Figure 14).
Figure 14. Structure of diacylated trehalose sulfate.
Acyl2SGL consists of an α,α-d-trehalose core esterifiedat the 2- and 3-positions with long-chain fatty acids andO-sulfated at the 2�-position. A number of sulfoglycolipidanalogues of natural acyl2SGL, incorporating a variety ofpolymethylated chiral fatty acids in place of the naturallyoccurring complex fatty acid moiety at the 3-position, weresynthesized and screened with the aim of identifying apromising candidate for the development of a new tubercu-losis vaccine. Although none of the synthetic compoundswas as potent as the natural sulfoglycolipid 45, some ofthese analogues showed promising capacities to activateT cells, determined by measurement of IFN-γ release.[82]
3.8. Bacillus anthracis
Bacillus anthracis is a spore-forming bacterium thatcauses anthrax in humans and in other mammals. Althoughantibiotic therapy is possible at an early stage of the disease,
L. Morelli, L. Poletti, L. LayMICROREVIEWgastro-intestinal and inhalation anthrax are often more re-sistant to treatment.[83] A major constituent of the outersurface layers of B. anthracis spores is a glycoprotein expos-ing multiple copies of the tetrasaccharide 46 (Figure 15).This tetrasaccharide includes an unusual monosaccharideresidue – 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucose, referred to as anthrose – at its non-reducing end.
Figure 15. Structure of the tetrasaccharide 46, a major componentof the outer surface layer of B. anthracis.
The tetrasaccharide 46 is unique to B. anthracis, not be-ing found even in closely related species, and therefore be-came a target for vaccine development. The first synthesisof the tetrasaccharide 46 was reported by Seeberger and co-workers, who used a convergent [2 + 2] approach(Scheme 5).[84] The terminal anthrose building block 50 wasprepared from d-fucose through inversion of the configura-tion at C-4 by triflation of the free hydroxy group followedby a SN2-type reaction with sodium azide. The other threebuilding blocks 47–49 were derived from l-rhamnose.
The tetrasaccharide 46 was then conjugated to KLH asa carrier protein by reductive amination and employed inimmunization studies. The conjugate induced tetrasaccha-
Scheme 5. Synthesis of the tetrasaccharide 46 by Seeberger et al.
ride-specific monoclonal IgG antibodies that bound exclu-sively to native B. anthracis endospores, indicating thatthese antibodies could serve as a sensitive and specific de-tection system for B. anthracis endospores.[85] The same au-thors also synthesized a pentasaccharide closely related to46.[86]
A second route to the B. anthracis tetrasaccharide wasdeveloped by the Kovác laboratory. It used a sequentialelongation strategy in which the reducing terminus was pro-vided with a spacer for protein conjugation.[87] More re-cently, the same authors described an improved and shorterroute to the target tetrasaccharide, based on a convergent[2 + 2] assembly. The major novelty of this approach lies inthe use of a disaccharide glycosyl donor containing the fullyassembled anthrose as one of the constituent sugar residues.Moreover, the synthesis is utilizable for gram-scale prepara-tion of the tetrasaccharide in conjugatable form.[88] Boonsand co-workers synthesized trisaccharide epitopes in whichthe anthrose moiety, generated from d-fucose, is N-acylatedin different ways.[89] When these trisaccharides were linkedto KLH, the conjugates were recognized by antisera raisedagainst spores of B. anthracis. Furthermore, it was foundthat the 2-O-methyl ether group of the anthrose residue iscritical for antispore antibody binding and that the 3-meth-ylbutyryl moiety is also important for the antigenicity ofthe oligosaccharide epitope.
The synthesis of the pentenyl glycoside of anthrax tetra-saccharide by a [3 + 1] approach in the Crich laboratorywas described. In this strategy, the construction of the 1,2-trans-glycosidic linkage in the terminal anthrose moiety wasachieved through the application of known α-nitrilium-ion-mediated β-selective glycosylation methodology,[90] whereasan iterative glycosylation strategy was used for the assemblyof the trirhamnan building block.[91] Moreover, a new,shorter route to the anthrose saccharide, utilizing inexpen-sive d-galactose as a precursor, was developed. In a similar
Synthetic Oligosaccharide Antigens for Vaccine Formulation
approach, Djedaïni-Pilard and co-workers prepared theanthrose monosaccharide in conjugatable form startingfrom d-galactose, demonstrating that this hapten is able toinduce a highly specific and sensitive immune response inrabbit when attached to a carrier protein.[92] O’Doherty’sgroup synthesized the anthrax tetrasaccharide 46 and ananalogue with an anomeric hexyl azide group by a de novostrategy from acetylfuran as a starting material(Scheme 6).[93]
Scheme 6. Retrosynthesis of the tetrasaccharide 46 from acetylfur-an by a de novo strategy.
The construction of the tetrasaccharide was achieved bya traditional [3 + 1] approach, in which the crucial stepswere Noyori reduction, Achmatowicz rearrangement,[94]
palladium-catalysed azide allylation, Luche reduction, anddiastereoselective dihydroxylation. An application of thisprotocol, illustrating the de novo preparation of theanthrose glycosyl donors 55 (trichloroacetimidate or di-phenylphosphate), is outlined in Scheme 7.
Additional carbohydrate antigens strictly associated toB. anthracis might offer exciting new targets for the devel-opment of improved vaccines against anthrax. Boons andco-workers, for example, recently synthesized the trisaccha-rides 64 and 65 (Figure 16), components of a unique poly-saccharide released from the vegetative cell wall of B. an-thracis.
It was found that sera from rabbits either exposed tospores of B. anthracis or immunized with polysaccharideconjugated to KLH recognize both the isolated polysaccha-ride and the synthetic compounds 64 and 65.[96] The See-
Scheme 7. De novo synthesis of anthrose glycosyl donors.
Figure 16. Structures of the synthetic trisaccharides 64 and 65 fromsecondary cell wall polysaccharide of B. anthracis.
berger laboratory reported the first total synthesis of thehexasaccharide repeating unit of the same polysaccha-ride.[97]
4. Antiviral Vaccines
Virus infections cause a great variety of diseases, rangingfrom the common cold to influenza, chronic hepatitis andlife-threatening AIDS (acquired immunodeficiency syn-drome). Throughout history, humankind has been devas-tated by viral epidemics. The 1918 influenza pandemickilled more than 20 million people worldwide;[98] nowadaysthe AIDS pandemic has killed over 25 million people. Someviruses have also been implicated in the onset of cancer inhumans. The prevalence and severity of many diseasescaused by viral infections justify tremendous efforts di-rected towards the development of antiviral drugs and vac-cines. During the 20th century, vaccines for many commonacute viral infections were developed and made widelyavailable. They have been most successful in cases in whichacute natural infection is self-limited and leads to long-last-
L. Morelli, L. Poletti, L. LayMICROREVIEWing protective immunity if the patient survives the initialinfection. In these cases, the best vaccine has usually beenthe one that most closely mimics the natural infection, suchas a live, attenuated virus. Indeed, a few years ago, a newlive attenuated influenza vaccine was licensed for intranasalaerosol administration.[99]
The development, however, of a vaccine that is effectiveagainst viruses that cause chronic infections, such as humanimmunodeficiency virus (HIV), hepatitis C virus (HCV),and human papillomavirus (HPV), might require consider-ation of a paradigm different from that described above. Avaccine that just mimics natural infection is not likely tobe adequate to induce protection. Moreover, there is muchconcern about the use of live attenuated viruses for vacci-nation against these diseases. These viruses have evolved toescape or evade the immune system, not to act as optimalvaccines. The challenge for the 21st century is to apply thelatest fundamental knowledge in molecular biology, virol-ogy and immunology to the development of vaccines thatare more effective than the natural infection in eliciting im-munity and are consequently effective against chronic viraland other infectious diseases, as well as cancer, that do notfit the classic paradigm.
Recent studies have shown that the glycoproteins ex-pressed on the surfaces of various viruses are stronglycorrelated with their virulence and immune evasion. Unlikebacteria or parasites, viruses can take advantage of hostglycosylation machinery to construct their own outer-sur-face glycans. Glycans are perhaps one of the most impor-tant classes of molecular components of cells and offer astill underappreciated diversity of structure and func-tion.[100] Much attention has been focused on study of N-glycosylation of viral proteins, because some viruses areshielded by dense layers of carbohydrates that serve criticalfunctions such as assisting protein folding, aiding entry intohost cells and evasion of detection by the immune system.Viruses co-opt host biosynthetic pathways to generate theirgenetic and structural material and use host glycosylationpathways to modify viral proteins. N-Glycosylation of viralenvelope proteins promotes proper folding through interac-tion with the host’s cellular chaperones and facilitatesproper trafficking through secretory apparatus. In additionto these “quality control” functions, changes in glycosyl-ation can reduce the ability of a host’s immune system torecognize a virus; HIV and influenza, for example, rely onexpression of specific oligosaccharides to evade detectionby the host immune system. In addition, N-glycosylationplays important roles in a diverse set of vital biologicalfunctions of viruses that are specific to various classes ofthese pathogens. The host-synthesized glycans are consid-ered to be immune-tolerated and thus enable viruses to es-cape immune surveillance. Moreover, the rapidly mutatingvirus genome can alter glycosylation sites and increase thestructural diversity of viruses. Because sugars borne onviruses are produced by host cells, they are similar to en-dogenous glycans and a clearly defined repertoire of viralglycan epitopes that can be targeted by vaccines is challeng-ing to identify. Despite these challenges, there have been
recent advances made in the development of carbohydrate-based antiviral vaccines, such as for HIV and influenzavirus. The ability to raise antibodies that can neutralize thevirus is one of the most effective forms of protection so farexploited in vaccines. Such antibodies have formed thebases for the poliovirus and influenza virus vaccines andthe major childhood vaccines that have been licensed foruse in the United States and elsewhere in the world.
4.1. HIV
AIDS was first described in 1981, as an outbreak of Pne-umocystis carinii pneumonia among homosexual men in theUSA.[101] Although AIDS was unrecognized as a diseaseentity 30 years ago, today UNAIDS (Joint United NationsProgramme on HIV and AIDS) estimates that 33.4 millionnow live with HIV type-1 infection, the etiologic cause ofAIDS, and that 2 million become newly diagnosed withHIV-1 each year (more than 15000 every day). Most ofthese new infections are sexually transmitted, but HIV isalso transmitted through contaminated blood or bloodproducts, or by use of contaminated needles or surgical in-struments.
HIV-1 is a highly mutagenic and variable virus from theRetroviridae family of viruses that contains multiple sub-types. There are two well-defined etiological agents ofAIDS: HIV-1[102] and HIV-2.[103] Both cause the disease,but HIV-1 appears to be more aggressive and spreads morerapidly.[104] Both retroviruses belong to the lentivirus sub-family. Lentiviruses produce characteristically slow, pro-gressive infections, in which the virus causes disease after along period of latency and persists in the host in spite ofthe host’s active immune response. A remarkable feature ofHIV is the dense carbohydrate (glycan) array that sur-rounds the exposed envelope antigens. In particular, thegp160 glycoprotein forms spikes at the surface of the virion(Figure 17).
Figure 17. HIV structure.
Gp160 is cleaved in the Golgi into two polypeptidechains: the transmembrane chain (gp41) and the externalchain (gp120). These remain linked together by noncovalentbonds. Gp41 anchors the glycoprotein spikes in the lipidbilayer of the envelope, maintains their trimeric organiza-tion[105] and plays a major role in fusion of the virus andcell membranes.[106] Gp120 carries the important antigenic
Synthetic Oligosaccharide Antigens for Vaccine Formulation
determinants of the virus and binds the CD4 receptor andCCR-5 or CXCR-4 co-receptors on the surface of targethelper T cells. The 120 kDa gp120 molecule contains about50 kDa of carbohydrates, consisting of a mixture of high-mannose and complex sialic acid-containing carbo-hydrates.[107]
As a result, the envelope protein (gp120) of HIV-1 is oneof the most heavily glycosylated proteins in nature, withmany of high mannose composition (Figure 18).
Figure 18. High mannose composition of the envelope proteingp120.
A recent survey of data from global HIV gp120 se-quences showed that the molecule had a range of possibleN-linked glycosylation sites of between 18 and 33 with amean of 25.[108]
However, the HIV genome encodes no gene productscapable of synthesizing carbohydrates: its surface antigensare glycosylated entirely by host cellular enzymes. This ex-tensive glycosylation is known to affect almost every aspectof virus biology, the transmission, and the nature of theimmune response to infection. The saccharide structures ex-pressed on the virus surface, however, are perceived as self-antigens, because they are synthesized by the host cell, andthis represents a major obstacle to the development ofcarbohydrate-based anti-HIV vaccines.
4.1.1. Carbohydrate-Based Anti-HIV Vaccines
The goal of an HIV-1 vaccine would be either to preventinfection or to reduce viral loads and clinical disease pro-gression after infection. An ideal vaccine would completelyblock infection and provide sterilizing immunity.
HIV-1 gp120 is a primary target for the development ofHIV vaccines. Immune response to the protein portion ofgp120 in infected individuals is not typically observed, be-cause of the extensive shielding of the conserved peptideepitopes by carbohydrates. The development of HIV vac-cines based on the carbohydrate epitopes of gp120 hastherefore become a natural choice. However, when consider-ing the use of carbohydrates in the development of HIVvaccines, two problematic issues arise. Firstly, as mentionedabove, the glycans exposed on the HIV envelope surface areimmunologically “self” molecules, leading to immunotoler-ance. Secondly, the combination of variability in the viralgenome and the microheterogeneity of N-glycans meansthat an extraordinarily high degree of HIV structural diver-sity exists. Therefore, to develop a potentially useful HIVvaccine, the important, but challenging, first step is to
identify defined, highly conserved and immunologicallyactive carbohydrate-containing epitopes in HIV gp120 aspotential antigenic targets.
Neutralizing antibodies are one of the main componentsof our immune response to pathogens. However, the rolethey have in the biology of HIV infection is not altogetherclear. Although there is limited immune response to HIV-1in vivo, a few neutralizing monoclonal antibodies (mAbs)that recognize conserved regions of the viral envelope havebeen isolated.[109] These include the neutralizing antibodies2F5 and 4E10, which target epitopes on the inner envelopeglycoprotein gp41,[110] and the neutralizing antibodies b12and 2G12, which recognize epitopes on the outer envelopeglycoprotein gp120.[111] The human monoclonal antibody2G12 is special among anti-HIV antibodies because it isuniquely capable of recognizing sugars on the immunologi-cally “silent” carbohydrate face of gp120 with high affin-ity[112] and of escaping immune tolerance. Calarese et al.[112]
solved X-ray crystal structures of Fab 2G12 and its com-plexes with the disaccharide Manα1�2Man and the high-mannose oligosaccharide Man9GlcNAc2. This study al-lowed the gp120–mAb 2G12 interaction to be charac-terized, highlighting the importance of the oligomannoseD1 and D3 arms in binding (Figure 18).[112,113]
Despite the fact that the carbohydrates adorning gp120are host-synthesized and can be considered “self”, the ab-normally dense clustering of oligomannoses might indicatethat the self carbohydrates are arranged in a “non-self”pattern, which in this case is a distinguishing immunogenictrait.[114]
Wang’s and Danishefsky’s labs independently undertookthe first step in synthetic carbohydrate-based HIV vaccinesand were among a few pioneering groups that set out toexplore the possibility of synthetic HIV vaccines based onthe 2G12 epitope.[109a,115] Wang assembled oligomannoseclusters on two distinct molecular scaffolds – cholic ac-id[109a] and d-galactose[115a] – to mimic the 2G12 epitope.The three Man9GlcNAc2 moieties, prepared from soybeanagglutinin by a reported procedure,[116] were attached to therigid scaffold 66 (Scheme 8), derived from cholic acid, re-sulting in the desired trivalent oligomannose cluster67.[109a,117] In a competitive inhibition assay, the half maxi-mal inhibitory concentration (IC50 = 21 μm) of the syntheticoligomannose cluster 67 showed a 46-fold increase in affin-ity for mAb 2G12 over Man9GlcNAc2Asn (IC50 = 13 μm, a73-fold increase over Man9GlcNAc2Asn). However, gp120binds mAb 2G12 in the nanomolar range. In another study,based on previous binding studies that had elucidated Man9
as the preferred subunit over other high-mannoses for 2G12recognition, Wang used ethyl α-d-galactopyranoside as amolecular scaffold to construct bi-, tri-, and tetravalentMan9 clusters (Scheme 9).[115a,117b,117c]
Monosaccharide-based scaffolds have several advantages,such as rigid ring structures and multiple functionalities.Moreover, they provide defined 3D spatial arrangements ofdifferent substituents. Although slightly increasedmAb 2G12 affinity was observed for the tetra-Man9 70(IC50 = 960 μm), it still could not reach the nanomolar
range observed for gp120. Evidently, the synthetic oligo-mannose cluster required optimization in glycan spacing orflexibility. Wang et al. also conjugated Man9-d-Gal clustersto the carrier protein KLH and a tetanus toxoid pept-ide.[115b] Immunization studies with the KLH conjugatedemonstrated that the glycoconjugate could induce moder-ate carbohydrate-specific antibodies in rabbits, which wereweakly cross-reactive to HIV-1 gp120.
Scheme 9. Structures of bi-Man9, tri-Man9, and tetra-Man9, together with synthesis of tetra-Man9 (70). PB = phosphate buffer.
In their most recent report, Wang et al. modified theirmolecular scaffold/linker in an attempt to eliminate linker-directed immune response, which suppressed the antibodyresponse to carbohydrate epitopes.[115c] They synthesized anew class of template-assembled oligomannose clusters byuse of a conformationally stabilized non-immunogenic cy-clic decapeptide (RAFT, Regioselectively AddressableFunctionalized Template)[118] as the template and the ter-minal-modified D1-Man4 arm oligosaccharide as the 2G12-recognizing subunits. RAFT consists of proline, glycine andlysine residues and provides two opposite functional faces(Figure 19) for the attachment of high-mannose moietiesand for the attachment of two T helper peptide epitopes.
Figure 19. RAFT-based D1-Man4 arm cluster.
The Man4 moiety was previously shown to exhibitmAb 2G12 binding comparable to that of its parent glycanMan9GlcNAc2,[119] and so it can be used as a functional yetsimplified analogue in HIV vaccine development. In view
Synthetic Oligosaccharide Antigens for Vaccine Formulation
of the weak immunogenicities of the previously synthesizedD1 arm clusters, the terminal mannose residue of the D1arm Man4 was selectively modified at C-6 with a fluorineatom. Accordingly, the 6-O-fluorinated Man4 glycoside 74was synthesized as outlined in Scheme 10.[115c]
Scheme 10. Synthesis of the 6-O-fluorinated Man4 glycoside 74.TMSOTf = trimethylsilyl trifluoromethanesulfonate; DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene.
In this way, a non-self glycan could behave as a hydro-gen-bonding acceptor, and moreover the unnatural modifi-cation was thought to enhance the immunogenicity. The oli-gomannose cluster 82 (Scheme 11) was efficiently assembledfrom the amino-functionalized RAFT 83 through CuI-cata-lysed 1,3-dipolar cycloaddition. Moreover, a T helper epi-tope peptide, to prime T cell activation, was conjugated tothe bottom face of RAFT.[115c] Animal immunization stud-ies using T helper-modified RAFT-based oligomannoseclusters to determine whether a strong carbohydrate-spe-
Scheme 11. Synthesis of the T-helper-modified RAFT-based oligo-mannose cluster 82. DCC = 1,3-dicyclohexylcarbodiimide; TFA =trifluoroacetic acid.
L. Morelli, L. Poletti, L. LayMICROREVIEWcific immune response can be generated are currently inprogress.
The Danishefsky laboratory has devoted a massive syn-thetic effort to the construction of HIV vaccines based onhigh-mannose and hybrid-type glycans of gp120.[115d–115f]
Scheme 12. Synthesis of high-mannose-type (100 and 101) and hybrid-type (104 and 105) glycopeptides. MCA = monochloroacetyl.
As well as the carbohydrate epitopes, their synthetic targetsalso included short peptide sequences of gp120 containingone of the key glycosylated asparagine residues (Asn-332).They employed chemical total synthesis in the construc-tion of gp120 fragments containing the high-mannose
Synthetic Oligosaccharide Antigens for Vaccine Formulation
(Man9GlcNAc2) and hybrid-type (Man5GalGlcNAc3) glyc-ans.[115e–115f,120] The syntheses both of high-mannose type(100, Scheme 12) and hybrid-type (104) glycopeptides wereoptimized by use of the convergent block ap-proach.[115e–115f] The monosaccharide building blocks 87–90 were assembled appropriately to afford the trisaccharideacceptor 91 and the donors 92–96. Suitable coupling reac-tions with this elaborated oligosaccharide blocks first pro-vided the free glycans 98 and 102, which were ultimatelyconverted into the glycopeptides 100 and 104, respectively,by condensation with the asparagine-containing peptide 97.Both the hybrid-type and the high-mannose-type glycopep-tides were studied in surface plasmon resonsnce (SPR)binding assays.[115d] The dimer resulting from disulfidebond formation in the synthetic high-mannose glycopenta-peptide exhibited substantial binding to mAb 2G12whereas the monomer showed significantly reduced bind-ing, thus providing additional evidence that multivalentbinding is operative in the gp120–mAb 2G12 interaction.The hybrid-type glycopentapeptide, whether in monomericor dimeric form, did not bind to mAb 2G12. This resultdemonstrates that hybrid glycans that lack the crucial D1arm present in high-mannose glycans are not recognized bymAb 2G12. Three pieces of evidence were elucidated: thepeptide motifs are not directly recognized by 2G12,[112] thehigh-mannose type is more likely to be recognized by 2G12,and, interestingly, when the sulfhydryl side chain of Cys331
was protected, binding was diminished. The last observa-tion supports the notion that structural motifs featuringmultiple glycans with suitable spatial orientations might becrucial for 2G12 binding, which is consistent with the crys-tal structure results reported by Wilson.[112]
With these structural criteria in mind, Danishefsky’sgroup constructed multivalent oligomannose motifs on acyclic peptide scaffold (Figure 20),[121] in analogy with themodular system designed by Dumy[122] and Robinson.[123]
In this system, which contains d-Pro-l-Pro sequences topromote β turns at both ends of the macrocycle,[124] posi-tions A–F should present side chains above the plane of themacrocycle. Placement of aspartate residues in any of thesepositions enabled attachment of the glycan Man9GlcNAc2
by Lansbury aspartylation.[125] Position G (with side chainprojecting from the opposite face of the scaffold) corre-
sponds to a cysteine residue, suitable for linkage to a carrierprotein. The cyclic peptide 106, containing two aspartateresidues, was prepared by automated solid-phase synthesisfrom prolinated trityl resin, followed by cleavage from theresin and a macrocyclization. After tert-butyl deprotectionof aspartate esters, the glycan Man9GlcNAc2 98 was at-tached by Lansbury aspartylation. The cyclic glycopeptidewas then coupled to the purified outer membrane proteincomplex (OMPC) derived from Neisseria meningitidis. TheOMPC is a macromolecular lipoprotein complex that servesas a highly effective immunostimulatory carrier for poorlyimmunogenic peptide and carbohydrate antigens.[126] Theseconjugates showed significant binding to mAb 2G12, al-though their affinities for mAb 2G12 remained weaker thanthose for gp120. Animal vaccination studies directedtowards evaluation of the synthetic conjugates as HIV vac-cines are currently underway.
Early work by Wong’s group was focused on the develop-ment of a synthetic methodology for the preparation andidentification of functional oligomannose derivatives. The“reactivity-based one-pot oligosaccharide synthesis” tech-nique[127] was employed, for example, in the preparation ofa series of oligomannoses corresponding to the D1, D2 andD3 arms of Man9.[119] Interestingly, simplified synthetic oli-gomannoses based on the D1 and/or D3 arms ofMan9GlcNAc2 were able to compete with gp120 in the in-teraction with mAb 2G12 better than Man9GlcNAc2 itselfin an ELISA assay. X-ray crystal structure studies of themolecules revealed that the D1 arm (107, 79% inhibition at2 mm, Figure 21) was the primary carbohydrate recognitionmotif for 2G12.
Man5 (108, 79% inhibition, Figure 21) also showed thesame inhibition level and can be viewed as a combinationof D2 and D3 arms containing a bivalent Manα1�2Manmotif. Independently, Seeberger and co-workers also foundthat the terminal Manα1�2Man unit was critical for 2G12recognition, but that a Manα1�2Man unit alone was notsufficient for antibody binding.[128] More recently, Wonget al. described further studies on the complexes ofmAb 2G12 with synthetic subunits of Man9GlcNAc2.[113]
Using the reactivity-based one-pot self-condensation re-action, they prepared the three previously undescribedManα1�2Man-containing oligomannose compounds 109–
111 (Man7, Man8 and Man9, respectively, Figure 21). Theydemonstrated that the carbohydrate specificity of 2G12 isless restrictive than originally believed.[112] 2G12 can bindto the Manα1�2Man at the termini of both the D1 andthe D3 arms of oligomannoses. Therefore, 2G12 could bindnot only to the D1 arms from two different N-linked oligo-mannoses on gp120, but also to both the D1 and the D3arms from different sugars in the oligomannose constel-lations on gp120. In another study, Wong et al. reported thesynthesis and biological evaluation of glycodendrons bear-ing Man4 and Man9 fragments.[129] Conceptually similar toWang’s and Danishefsky’s proposals, Wong’s candidateswere based on multivalent presentation of glycans. InWong’s glycodendrons, variably branched/sized AB3-typedendrimers were decorated with Man4 and Man9, by meansof copper(I)-catalysed cycloadditions between the terminalalkynes of the dendrimer and Man4 and Man9 azides. Bind-ing studies indicated that the second-generation Man9 den-dron, displaying an average of nine glycans, was the mostpromising candidate for vaccine development, because it ex-hibited significant inhibition both of the gp120–mAb 2G12and of the gp120–DC-SIGN interactions.[130]
The enhanced 2G12 complex avidity came from themultivalent nature of Man9. Glycodendrons can thus beexplored both as carbohydrate vaccine candidates and asantiviral therapeutic agents, preventing sexual transmissionof HIV. However glycodendrons bind to mAb 2G12 only atμm concentrations, and Man9 and the synthetic D1 arm
(Man4) oligomannose mimics of the natural Man9GlcNAc2
are the best subunits for 2G12 recognition.The distance between the two primary binding sites in
2G12 is 30 Å,[112] and no more systematic investigation ofthe optimal spacing geometry between the ligands to max-imize the cooperativity has yet been reported. To this end,in 2009 Winssinger and co-workers reported the first exam-ples of PNA-encoded oligosaccharides that mimic the 2G12epitope.[131] They designed a pilot library of over 30 archi-tectures, but significant binding (μm) was only observed forconjugates containing two Manα1�2Man units. This ex-ample illustrates the importance of multimeric recognitionwith controlled topology.
Recently, a Novartis research group exploited the flexiblepolyamidoamine (PAMAM)[132] scaffold to generate four-and eight-valent sugar-dendrons of HIV-1-related oligom-annose antigens.[133] Astronomo and co-workers, on theother hand, tried to increase the affinity to monoclonal an-tibody 2G12 using oligomannoses linked to icosahedralvirus capside scaffolds as immunogens.[134] The icosahedralparticles display reactive amines on their surfaces. Glycanscan be attached to these in different numbers, at appropri-ate distances and with conformational flexibility and dif-ferent geometrical arrangements for interaction with 2G12.The bacteriophage Qβ capside glycoconjugates presentedoligomannose clusters that bind the antibody 2G12 withhigh but still insufficient affinities.
Very recently, Davis and co-workers designed and syn-thesized a non-self sugar mimic of the HIV glycan shield,obtaining interesting results.[135] They focused on a uniquenon-self D1 arm mimic and found that this sugar scaffold,both as an isolated sugar and as a glycoconjugate, displaysbetter inhibition of 2G12/gp120 binding than the naturalD1 arm. Their strategy was based on the observation that,in a panel of monosaccharides, d-fructose is a better inhibi-tor of 2G12–gp120 binding complex than mannose. Thestructure of fructose, when rotated by 180°, is similar tothat of mannose, and differs only at C-1 and C-5. d-Fruc-tose can also potentially enter into additional hydrogenbonding interactions in relation to mannose (Figure 22).
Figure 22. Comparison between d-fructose and d-mannose struc-tures.
From the crystal structure of Fab 2G12 in complexationwith d-fructose they were able to determine: a) that the d-fructose adopts the pyranose form, resembling d-manno-pyranose in the 2G12 binding site, b) that d-fructose forms
Synthetic Oligosaccharide Antigens for Vaccine Formulation
the same contacts with the 2G12 binding site as are madeby the terminal mannoside in Manα1�2Man, and c) thatthere are additional direct H-bonds and also water-medi-ated interactions involving the C-5 substituent of fructose.No other simple sugars or mannose disaccharides withother linkages inhibit 2G12–gp120 binding, and this en-hanced binding affinity could lead to the design of a hybridnon-self sugar.
On the basis of modelling results, they designed a targetpanel of non-self sugars, containing R substitutions andmodifications at the C-3, C-5 and C-6 positions of the ter-minal sugar of the D1 arm (Figure 23). If R is not a largegroup the manipulations are tolerated by 2G12 and are be-lieved to enhance the binding. By starting from d-mannose,through oxidation and addition procedures, the monosac-charides 112, 113, 114 and 115 were synthesized for evalu-ation by ELISA, in order to determine the best candidatefor introduction into the D1 arm, as well as the ability ofthese fructose-like monomers to inhibit binding of 2G12 togp120. From the panel, the C-6 methyl-substituted com-pound 115 emerged with an IC50 value more potent thand-mannose and also more potent than d-fructose. The fruc-tose-like monosaccharides 113a, 113b, 114 and 115 werethen incorporated at the terminus of the D1 arm.
The terminal non-self monosaccharides were added byuse of the corresponding perbenzylated thiophenyl glycosyldonor and dimethyl(methylthio)sulfonium triflate(DMTST) as activating agent, and non-self D1-arm sugarswere obtained after deprotection (Figure 24). Compound116 not only binds better than the other non-self variants,but it is the only nonself D1 arm derivative to show betterinhibition (IC50 value four times lower) of 2G12–gp120binding than the natural D1 arm and is the most potent
monovalent 2G12 ligand known. They lastly investigatedthe immunogenicity of the glycan shield mimetic Qβ-conju-gated D1 arm mimic 117, generated by coupling of 116 witha virus-like particle Qβ through Huisgen cycloaddition, rel-ative to the corresponding Qβ-conjugated natural D1 arm118.[134,136] The conjugates 117 and 118 bound 2G12 withnanomolar affinities, but also in this case the elicited anti-bodies do not neutralize HIV, possibly because of an incor-rect presentation mode of the glycan on the conjugates.
Figure 24. Structures of the nonself mimetic 116, of the Qβ-D1 armglycan 118 and of the Qβ-nonself mimetic 117.
Overall, suitable optimization of glycan spacing, flexibil-ity and immunogenicity are required for synthetic oligo-mannose compounds to induce 2G12-like neutralizing anti-body sufficiently. In the absence of an effective vaccine forHIV/AIDS, microbicidal lectins are promising comple-ments to traditional barrier protection.
4.2. Influenza Virus
The influenza viruses belong to the Orthomyxoviridaefamily, which is subdivided into three serologically distincttypes: A, B and C. Only influenza viruses A and B appearto be of concern as human pathogens, whereas influenza Cvirus does not seem to cause significant disease.[137] More-over, of the three virus classes, influenza A is notorious forcausing major epidemics and pandemics. Influenza Aviruses are responsible for causing seasonal epidemics andcaused the three pandemics that occurred in the 20th cen-tury (1918, 1957 and 1968), as well as the 2009 H1N1 pan-demic, the first pandemic of the 21st century. Human influ-enza is predominantly an upper respiratory tract infectionthat is typically mild and self-limiting, although sometimessevere complications and death can occur.
The enveloped viruses and some non-enveloped viruseshave carbohydrates as major surface constituents. For along time the biological functions of these carbohydrateswere obscure, but there is now increasing evidence that theyplay some important roles in the life cycles of these viruses.The influenza A viral envelope contains two extensively gly-cosylated surface proteins: hemagglutinin (HA) and neur-aminidase (NA). Both glycoproteins function by recogniz-ing N-acetylneuraminic acid (Neu5Ac, also called sialic
L. Morelli, L. Poletti, L. LayMICROREVIEWacid) molecules on the host cell. The virus serotypes arenamed according to HA and NA glycoproteins: to date, 16HA and nine NA types have been described, but threemajor HAs (H1, H2 and H3) and two NAs (N1 and N2)account for influenza pandemics in humans, resulting in theemergence of pandemic strains H1 in 1918, H2 in 1957, andH3 in 1968. Recently, three small outbreaks have arisenfrom avian subtypes (H5, H7 and H9) that managed tomake direct leaps to humans, but their low transmissibilitiesprevented major new epidemics.[138] However, the emer-gence of future influenza virus pandemic strains is likely,[139]
and their severity will depend on the ability to contain andcombat infection by timely development of an appropriatevaccine.
HA is made up of three identical subunits and is an-chored to the lipid membrane of the virus (Figure 25).[137b]
This glycoprotein seems to have two significant roles.Firstly, it provides an initial point of contact (the globular“head”) for the virus to the target host cell-surface glyco-conjugates through α-ketosidically linked terminal Neu5Acresidues.[140] The second key role of HA is to trigger theinternalization process of the virus through fusion of theviral envelope with the host cell.[140a,141] A multitude of in-fluenza virus HA structures have been determined, as wellas the structures of several HA–ligand complexes.[142]
Figure 25. Structure of influenza virus and interaction with hostcell surface.
Oligosaccharide side chains are distributed all over thesurfaces of the glycoprotein spikes. The HA mediates entryof the virus into the cell by binding to receptors and endo-cytosis, followed by fusion of the viral envelope with themembrane of the endosome. In order to achieve fusion, theHA has to be activated through a multi-step process con-sisting of cleavage into the amino-terminal fragment HA1and the carboxy-terminal fragment HA2,[143] followed by aconformational change that occurs at low pH. As a result,the fusion peptide, which is close to the amino terminus of
HA2, is exposed in such a way that fusion can occur.Whereas HA binds to sialylated receptors on the host toinitiate virus infection, NA hydrolyses sialic acid residuesfrom host cell receptors to assist in the release of progenyvirus that go on to infect neighbouring cells.[144] The func-tional interplay between HA and NA in virus attachment/release is dependent on the glycans of HA.[145] Overall, thecarbohydrates in HA and NA serve a variety of purposes,including assistance in protein folding, intracellular trans-port, receptor binding, virus release, infection, immune eva-sion and neurovirulence.[146]
The number of glycosylation sites in HA ranges from fiveto 11, and several glycosylation sites are conserved in anarray of animal and human influenza A viruses.[147] Inmany virus subtypes, however, selection has resulted in theintroduction of additional glycosylation sites over time.[148]
Because the glycans of the HA have been clearly implicatedin immune evasion[146f–146i,149] this global increase/variationin glycosylation sites is believed to generate antigenic vari-ants, so the concept of an evolving or dynamic glycan shieldapplies here. On the other hand, HA also contains severalregions that have always lacked carbohydrates, and site-spe-cific mutagenesis experiments demonstrated that incorpo-rating glycans into these regions resulted in the disruptionof HA transport to the cell surface.[150]
The role of HA carbohydrates in host cell attachmentand release has been studied in detail;[146b,151] optimal virusreplication relies on a delicate functional balance betweenthe surface proteins HA and NA, and the glycosylationstate of HA has a direct impact on the balance.[137b] Thecarbohydrates associated with influenza virus surface pro-teins are therefore deeply involved in many viral functions,and their presence can result in either positive or damagingeffects depending on location.
4.2.1. Carbohydrate-Based Anti-Influenza Vaccines
The seasonal influenza vaccines currently availablemainly consist of purified HA/NA blends and are effectivefor many people. However, the immunogenicity is reducedfor high-risk populations: some studies, for example, haveshown that vaccination of the elderly reduced influenzacontraction by only 50 %.[152] Research focusing on the ex-tensively glycosylated HA of virulent strains is therefore ur-gently needed.
Galili and co-workers designed a carbohydrate-basedstrategy for improving the immunogenicity of influenza vac-cine by exploiting the mechanism of antibody-dependentantigen uptake.[153] In particular, the naturally occurringanti-Gal antibody is the most abundant antibody in hu-mans,[154] and moreover it is the only human antibody thatcan target antigens to APCs. As a consequence, any particu-late or soluble antigen that has α-Gal epitopes (Galα1–3Galβ1–4GlcNAc-R) will form immune complexes withanti-Gal antibodies and will be targeted for effective uptakeby APCs. The modified influenza vaccines explored by Gal-ili harness these special properties of the α-Gal epitope andanti-Gal.[155] They incorporated the α-Gal epitope into theN-glycans of HA by an enzyme strategy[156] and they hy-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
pothesized that the synthesis of α-Gal epitopes on inacti-vated influenza virus and subsequent immunization with in-activated influenza virus expressing α-Gal epitopes shouldresult in a significantly higher level of immune responsethan that measured after immunization with unprocessedinactivated influenza virus that lacks α-Gal epitopes. Thisstrategy was tested with α-1,3-galactosyltransferase (α-1,3GT) knockout mice, which effectively mimic the humanimmune system in this context, and the PR8 influenza virusstrain.[156] Modification of the PR8 N-glycans by recombi-nant α-1,3GT produced the influenza virus strain contain-ing the α-Gal epitope (PR8αGal), which was used as a modi-fied influenza vaccine. Mice immunized with PR8αGal pro-duced greater quantities of PR8-specific CTL and Th cellsand higher titres of anti-PR8 antibodies than mice immu-nized with PR8 lacking α-Gal epitopes. The expression ofα-Gal epitopes on surface glycoproteins, to enhance im-mune response to influenza virus vaccines, is a very attract-ive strategy because it could also be applied to improve theimmunogenicities of HIV gp120[157] and tumour vac-cines.[158]
Wong and co-workers recently discovered that systematicsimplification of the N-glycans on HA resulted in an in-crease in binding affinity to α-2,3-receptor sialosides butnot to α-2,6-receptor sialosides.[159] Because the viral trans-mission begins with a critical interaction between HAglycoprotein, which is on the viral coat of influenza, andglycans containing sialic acid (SA), which are on the hostcell surface, various defined HA glycoforms were prepared[from fully glycosylated HA complex type (HAfg) to desial-yated complex type (HAds) and from high-mannose type(HAhm) to monoglycosylated HA (HAmg)] in order to eluci-date the role of HA glycosylation in this important interac-tion, and their binding affinities and specificities werestudied by means of a synthetic SA microarray. This is anot-previously-described study to show the effect of HA’souter and inner glycans on receptor binding and to dissectthe binding affinities and energetic contributions of HA–receptor interactions quantitatively. Because HA withtruncated glycans can recognize α-2,3 receptor sialosideswith higher binding affinities and less specificity, reductionof the lengths of glycans on HA might increase the risk ofinfection. HA with a single GlcNAc (HAmg) retained theintact secondary structure in relation to the HAfg. More-over, HAmg antiserum showed stronger neutralization andwas much more protective than HAfg vaccination in a lethaldose of H5N1 challenge study. Overall, HAmg is a promis-ing vaccine candidate for influenza because it exposes theconserved peptide epitopes that are much more immuno-genic but were originally hidden by massive glycans. Al-though it is unclear how the changes in HA–receptor inter-action arising from glycosylation affect the infectivity of thevirus and the NA activity in the viral life cycle, this strategyoffers a new direction for vaccine design and, together withother different vaccine strategies[160] and recent discoveriesof HA-neutralizing antibodies,[161] should facilitate the de-velopment of vaccines against viruses such as influenza,hepatitis C virus and HIV.
A parasite can be broadly defined as any organism thatlives on or in another organism while actively harming thelatter. By this definition parasites include viruses, bacteria,protozoans, and helminths (worms). However, in the ver-nacular, the term “parasite” is used exclusively to describeinfectious protozoans and helminths.
Although infections by such organisms are a majorworldwide health concern, no vaccines exist for the majorhuman parasitic diseases, such as malaria, African trypano-somiasis, leishmaniasis and schistosomiasis. In most casescarbohydrates represent the dominant antigens of these in-fections, but their use as immunogens and as potential vac-cine candidates has been very limited by the technical diffi-culties involved in obtaining large quantities of parasitesand the historical difficulties in defining structures of un-usual parasite-derived glycan antigens.
Parasites differ significantly from bacteria in their struc-tures, modes of infection and defence mechanisms againsthost immune attack.
They have evolved a myriad of strategies to exploit andevade immune killing, such as residing intracellularly inhost tissues, or techniques of molecular evasion achieved byalterations in expression levels of antigens during parasitedevelopment in the infected host. Thus, unlike bacterial in-fections, parasitic infections tend to be chronic and long-lived.
These pathogens display tremendous differences in struc-ture, morphology and size, ranging from 14–33 μm in theTrypanosoma parasite to 6000–14000 μm in the Schisto-soma helminth. This makes adult worms relatively refrac-tory to immune lysis in infected individuals because –whereas immune cells might be able to bind and recognizebacteria and protozoans – APCs will never phagocytize andprocess large, intact helminths, even though they displaymost of their glycan antigens on their surfaces.[162] Thus,although infection generates immune responses directedtowards the glycan antigens, such responses and antibodytitres are probably not sufficient to clear an existing infec-tion. More effective helminthic antigens are likely to origi-nate from secreted products, in which glycoconjugates con-tain the antigenic determinants that are most likely to beshared between secreted and cell surface molecules. As analternative, effective vaccines against parasitic helminthscould be successful if targeted on the early infectious stageswhen the parasites are most sensitive to immune attack anddestruction.
The complex life cycles of parasites and the pathologiesthey cause provide different avenues for the design of vac-cine strategies to limit their propagation, transmission andpathology.[163] A conventional subunit type vaccine couldbe made to induce an immune response that directly recog-nizes and kills a parasite in a definitive host. Alternativestrategies could include transmission blocking vaccines,which would interfere with parasite development in inter-mediate hosts, and anti-pathogenesis vaccines, which wouldblock the effects of toxins or other pathogenic parasite-de-
L. Morelli, L. Poletti, L. LayMICROREVIEWrived molecules without killing the parasite directly (clinicalimmunity).
5.1. Infection by Plasmodium falciparum
Plasmodium falciparum is a protozoan parasite thatcauses malaria, an infectious disease threatening half of theworld’s population. In 2008 it was responsible for 243 mil-lion estimated cases and nearly 863000 deaths.[164] The com-plex lifecycle of the parasite, the differentiation of its anti-gens and the protective immune mechanisms at each stagehamper the development of efficient preventative strategies,so insecticide devices are still the main tools to prevent ma-laria.[10,165]
Over 90% of the total glycoconjugates made by malariaparasite are GPI anchors,[166] which appear to be dominantmalarial toxins responsible for many of the severe patholog-ical consequences of the disease[167] and so can be regardedas a potential targets for anti-toxic vaccines for clinical im-munity.
In 2002, Seeberger’s group synthesized the nonacylated,nontoxic P. falciparum GPI glycan 119 (Figure 26) andtested its immunogenicity in rodents after conjugation withOVA and KLH. Remarkably, 60–75% of the mice immu-nized with 119 were substantially protected from deathcaused by malaria and survived, compared with 0–9 % sur-vival rates for unvaccinated mice.[168]
The need for gram amounts of antigen for preclinical andclinical studies focused attention on synthetic methods suit-able for easy scale-up. The native presentation of the anti-gen through the 2-Ino position was desirable for better vac-cination, which prompted the development of a more gene-ral and practical synthetic strategy, based on building-block
Figure 27. GPI fragments synthesized by Seeberger et al.
assembly and leading to GPI derivatives such as 126 (Fig-ure 27).[169] Together with 126, a series of GPI derivativeswere synthesized (compounds 120–125, Figure 27). These
Figure 26. Structure of natural and synthetic P. falciparum GPIstructure.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Scheme 13. Synthesis of the heptasaccharide 125. TCA = trichloroacetimidate, Piv = pivaloyl, TIPS = triisopropylsilyl.
differed in the number of mannose moieties and in the pres-ence of an aminoethyl phosphodiester group at the Man3
6-O position, displayed by the natural toxin.[170]
The synthetic targets required the adoption of a strategybased on late-stage coupling and modification steps, withuse of benzyl moieties as permanent protecting groups andan azido group as latent amino functionality, so that theirfinal removal could be achieved through Birch reduction.The installation of the phosphodiester groups was achievedby use of H-phosphonate chemistry. The key point of thesynthesis was the use of the mannose building blocks 128–130 and the known pseudodisaccharide acceptor 131 in aconvergent [n + 2] coupling strategy, as depicted inScheme 13.
Compounds 120–127 were covalently bound in picomo-lar amounts on the surfaces of glass sides, providing a plat-form for parallel screening of multiple sera on singleslides.[171] Initial studies showed a length-dependent bindingof the GPI glycan to the malaria-exposed sera, with apentasaccharide representing the minimal epitope for natu-rally elicited anti-GPI antibodies, whereas the results weredivergent with respect to the presence of the Man3 phos-phate ethanolamine group. The Man4 moiety showed a cru-cial role in displaying a broader anti-GPI response, proba-bly involving some nonmalarial species, whereas the Man3–GPI response might represent a more specific anti malarialGPI response.
The same microarray was used to investigate the age de-pendence of the development of humoral immune responseagainst GPI in malaria-exposed children under the age of18 months,[172] revealing that sera from subjects with severemalaria and from healthy children contained antibodiesthat predominantly recognise synthetic Man3–GPI andMan4–GPI. In contrast, antibodies in sera from childrenwith mild malaria also showed substantial reactivity with
truncated glycans consisting of glucosamine-inositol moie-ties without mannose or with only one or two mannoseresidues (compounds 120–122 in Figure 27).
5.2. Infection by Leishmania spp.
Leishmania are a genus of sandfly-transmitted protozoanparasites that cause a spectrum of debilitating and oftenfatal diseases in humans throughout the tropics and sub-tropics. One of the survival strategies in the Leishmania lifecycle involves the formation of an elaborate and dense cell-surface glycocalyx composed of glycoconjugates,[173] in par-ticular phosphoglycans, essential for the virulence of theparasite.[174]
The intriguing structure of the leishmanian lipophos-phoglycan (LPG, Figure 28) consists of four distinct func-tional domains: the alkylisophosphatidylinositol lipid an-chor, the conserved glycan core with an internal Galf resi-due, variable phosphoglycan (PG) repeats and a neutral oli-gomannose cap. The most distinct feature is the PG do-main, made up of [�6)Gal-β-(1�4)-Man-α-1-phosphate]nrepeats (n = 2–32), which is unique among eukaryoticcarbohydrates, as well as the rare Gal-β-d-(1�4)-Man con-nection. Structure–activity relationships have been pro-posed for each of the domains.[175] Although LPG is ex-pressed by all Leishmania species, their structures are re-markably stage- and species-specific.[24]
Notable work, starting in the 1990s, was carried out byNikolaev et al., in the synthesis of a series of Leishmaniaphosphoglycans (some examples are compounds 140–144ain Figure 29) with the aim of elucidating the biosyntheticpathway leading to the construction of the backbone and ofthe branches. The synthetic strategies they used, extensivelydescribed in a recent review by the same author,[24] ranged
L. Morelli, L. Poletti, L. LayMICROREVIEW
Figure 28. Structure of the natural Leishmania phosphoglycan.
from blockwise chain elongation to stepwise chain elong-ation – both in solution and in MPEG phase – and to thepolycondensation approach.[176] The key step for the intro-duction of the phosphate bridge between the repeating unitswas H-phosphonate coupling, in which an anomeric H-phosphonate is activated to form a mixed anhydride with asterically hindered carbonyl chloride (e.g., adamantyl chlo-ride or pivaloyl chloride), followed by esterification with theappropriate alcohol and oxidation with iodine and aqueouspyridine.
The same group also exploited these well established syn-thetic methods to prepare a series of synthetic PGs fromL. donovani, L. mexicana and L. major with the aim of elu-cidating their immunogenical properties.[177] These com-pounds were coupled to tetanus toxin fragment C (tetC)[178]
and used to immunize BALB/c mice and infected both bythe bite of L. mexicana-infected sand flies and by subcuta-neous needle inoculation of the L. mexicana infectingagent.[179]
Immunization with Leishmania mexicana promastigotesecretory gel (PSG) or with the chemically defined syntheticglycovaccines containing the glycans found in L. mexicanaPSG (Figure 29) was capable of conferring significant pro-tection against challenge by the bite of infected sand flies,providing a new target for Leishmania vaccine development.
The Dundee group also prepared the C-phosphonate an-alogues of the [�6)Galp-β-(1�4)-Man–α-1-phosphate]n re-peating unit constituting the PG backbone common to allLeishmania species. The phospho-disaccharide moietieswere assembled in a blockwise manner starting from thebuilding blocks 146–148, available from a common precur-sor, the known phosphonate 145 (Scheme 14).[180] The C-phosphonate groups connecting the repeating units of thePGs, lacking the anomeric oxygen, represent a promisingclass of mimics of the phosphodiester bridge, because theyconserve the steric properties of the natural compounds butare resistant to hydrolysis by acids or enzymes.[181]
Vishwakarma’s group found an elegant synthetic way toprepare the repeating unit disaccharide of Leishmania PGby starting from lactal, thus avoiding the difficult β-(1�4)
glycosylation step in the construction of the chain. The keyfeatures of the strategy are a) the gluco-manno transforma-tion by glycal chemistry, and b) the regioselective 1-O-de-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Scheme 14. Synthesis of phosphono analogues of Leishmania PGs. Dec = decenyl.
acetylation for the introduction of the phosphate by the H-phosphonate method.[182] This strategy showed great versa-tility, allowing the selective protection of the 6�-O-positionof lactal with a tert-butyl dimethylsilyl ether (TBS) and theelongation of the chain both from the nonreducing andfrom the reducing end. In this way, linear dimers and tri-mers of the phosphodisaccharide were obtained by iterationof simple deprotection steps and coupling by H-phos-phonate chemistry (Scheme 15).[183]
The methodology was notably applied to the solid-sup-port synthesis of di-, tetra- and hexasaccharide repeatingunits, accomplished on Merrifield resin. The specific prob-
lem of the lability of the anomeric phosphodiester betweenthe PG repeating units was circumvented by the introduc-tion of the novel cis-allyloxy linker, which, after a carefulscreening of cleavage conditions, allowed a successful un-loading of the PG oligomers from the resin without degra-dation of the phosphodiester functionalities (Scheme 16,a).[184]
Scheme 16. Solid-phase and polycondensation strategies in the syn-thesis of Leishmania PG oligomers. TEAB = triethylammoniumhydrogen carbonate buffer; TBS = texyl dimethylsilyl; Piv = pival-oyl.
L. Morelli, L. Poletti, L. LayMICROREVIEWThe New Delhi group also successfully exploited the
polycondensation approach to the construction of the PGchain, starting from the key building block 158(Scheme 16b). The progress of the polycondensation reac-tion could be easily monitored by in situ 31P NMR spec-troscopy. The size of the products 159 was determined bytwo independent methods based on negative ion ESMS and31P NMR analysis, revealing a mixture containing 19–22phosphoglycan repeats.
Finally, Seeberger’s group prepared the tetrasaccharidecap of LPG (Figure 28) both in solution and on solidphase.[185] Their versatile strategy was based on the use ofa pentenyl glycoside as the solid-phase linker and the or-thogonally protected mannosyl building block 160(Scheme 17) as the reducing end. The synthetic cap tetra-
Scheme 17. Solid-phase synthesis of Leishmania PG oligomers withthe aid of a pentenyl glycoside linker. TDS = texyl dimethylsilyl;TCA = trichloroacetimidate.
Scheme 18. Synthesis of glycoconjugates of Leishmania PG cap tetrasaccharide.
saccharide was conjugated to a phospholipid, to the influ-enza virus coat protein HA and to the carrier protein KLH(compounds 165–167, Scheme 18), to form semi-syntheticvaccines.[186]
Constructs 166–167 were embedded into the lipid mem-branes of reconstituted influenza virus virosomes, actingboth as adjuvants and as carriers. Virosomial formulationswere used to immunize BALB/c mice intramuscularly andwere able to elicit both IgM and IgG anti-glycan antibodies.The antisera cross-reacted in vitro with the correspondingnatural carbohydrate antigens expressed in Leishmania cells,demonstrating the potential vaccine activities of these twoglycoconjugate antigens.
5.3. Infection by Tripanosoma cruzi
The protozoan parasite Trypanosoma cruzi is a causativeagent of Chagas’ disease, which affects about 18 million in-dividuals in South and Central America, leading to about21000 deaths each year.[187] Hematophagous reduviid vec-tors, infesting the houses of poor people, transmit the para-site to vertebrates by leaving parasite-containing faeces af-ter taking a bloodmeal. Throughout the life cycle, T. cruziproduces both common and stage-specific GPI-anchoredcell-surface macromolecules. Local release of GPI-anchoredmucins by the bloodstream trypomastigote stage of theparasite is believed to be responsible for the developmentof parasite-elicited inflammation, which causes cardiac andother pathologies associated with the acute and chronicphases of Chagas’ disease,[188] suggesting that these mucinsare potential vaccine targets for clinical immunity.
Although some works reporting the synthesis of carbo-hydrate substrates for enzymes involved in the biosynthesisof T. cruzi GPI have been published,[189] few papers describ-ing the preparation of carbohydrate components for poten-tial anti-Tripanosoma vaccines have appeared.
Konradsson et al. reported the synthesis of the heptasac-charidyl myoinositol 168 (Figure 30), the inositolphos-phoglycan of the T. cruzi glycoinositolphospholipid(GIPL), containing the unusual, parasite-specific 2-amino-ethyl phosphonic acid at C-6 of the nonacylated GlcNp resi-due.[190] This glycan, attached to a ceramide lipid anchor,is a major cell membrane constituent in the proliferativeepimastigote stage of T. cruzi. The synthesis took advantage
Synthetic Oligosaccharide Antigens for Vaccine Formulation
of a previous work by the same group, describing the syn-thesis of the disaccharide 172 (Scheme 19).[191] This was in-troduced at the end of the synthesis, due to the presence ofa diastereoisomeric mixture at the phosphorus atoms andto its difficult, multi-step preparation. It was successfullycoupled to the hexasaccharide intermediate 169 throughDMTST activation of the thioethyl glycoside, providing theprotected octasaccharide 173. The deprotection sequenceleading to the final compound required the removal of theacetals as the final step, in order to avoid phosphate mi-gration and complex product formation.
Figure 30. Inositolphosphoglycan of the T. cruzi GIPL.
Nikolaev et al. described the synthesis of compounds174a and 174b (Scheme 20), components of the GPI frac-tion of T. cruzi trypomastigote mucins. Their extraordinaryproinflammatory activities, comparable to those of bacte-rial lipopolysaccharides, are probably due to the presenceof unsaturated fatty acids in the sn2 position of the alkyl-acylglycerolphosphate moiety and/or with d-Gal branchesalong the glycan core.
The synthetic pathway followed for their synthesis hadsome key features: 1) the exploration of benzoic esters andacid-labile permanent protecting groups (acetals and N-Boc), as a result of the presence of unsaturated fatty acids,which hampered the use of benzyl ethers, 2) the use of or-thogonal silyl ethers as blocking groups for O-6 in d-GlcN(triethylsilyl, TES), O-6 in d-Man-3 (primary tert-butyl di-
Scheme 20. Retrosynthesis of compounds 174, components of the GPI fraction of T. cruzi trypomastigote mucins. TES = triethyl silyl;TBS = tert-butyldimethylsilyl; SEM = 2-trimethylsilylethoxymethoxy.
methylsilyl, TBS), and O-1 in myoIno (secondary TBS), toensure further introduction of the P-containing esters, and3) a mild basic treatment in a polar solvent for the finaldeprotection, expected to cleave the benzoates preferentiallyand to leave the fatty acid ester of the lipid mostly intact asa result of micelle formation.
The protected hexasaccharide 175 was subjected to aseries of desilylation/phosphorylation steps leading to theprogressive introduction of the phosphates. Final removal
L. Morelli, L. Poletti, L. LayMICROREVIEWof the permanent protecting groups led to the target com-pounds. Preliminary experiments on the hexasaccharides174 with TLR-transfected Chinese hamster ovary (CHO)cell lines showed they stimulated TLR2-transfected cellsand not TLR4-transfected cells, like naturally occurringtGPI.[192]
5.4. Infection by Toxoplasma gondii
Toxoplasma gondii is a ubiquitous protozoan parasitethat is the cause of toxoplasmosis, a benign and asymptom-atic infection that can be controlled by the immune systemsof immunocompetent people. Among AIDS patients, how-ever, it is a major cause of death, and primordial infectionduring pregnancy can cause congenital toxoplasmosis withthe risk of severe infection of the foetus.
A carbohydrate-containing low-molecular-mass antigenhas been reported to exhibit immunological characteristicssuitable for serological diagnosis of acute toxoplasmosis.[193]
This antigen was identified as a family of protein-free GPIglycolipids, and recently the structures of these GPIs wereelucidated and shown to elicit an early immune response inhumans.[194]
Both lipid-free and lipid-containing GPI anchors fromT. gondii were synthesized by two groups, exploiting suitableprotecting group patterns a) for the key mannose monomerc, located at the branching position, and b) for the intro-duction of two different phosphate diesters at C-1 in themyoIno moiety and at C-6 in the nonreducing Man moiety.
Scheme 21 shows the building blocks used bySchmidt[195] and Seeberger[196] in the synthesis of the GPIderivatives 180a–c. Biological analysis of the compounds bySchmidt et al. revealed their ability to activate macrophages.Moreover, non-lipidated GPI was able to induce TNFα pro-duction, the same effect produced by protozoan GPIs, as
Scheme 21. Building block strategy in the synthesis of the Toxoplasma GPI anchors 180a–c. MPM = p-methoxyphenyl; PMB = p-methoxybenzyl; TBDPS = tert-butyldiphenylsilyl; TIPS = triisopropylsilyl; TCA = trichloroacetimidate.
well as to activate TLR2- and TLR4-expressing cells, sug-gesting their role in the development of innate and adaptiveprotection from T. gondii parasites.[197]
5.5 Infection by Helminths
As mentioned above, an effective immune response tohelminth infections can be promoted by antigens originat-ing from products secreted by the parasites, or by early-stage carbohydrate antigens.
A few examples describing the preparation of anti-hel-minth vaccine candidates have been reported in the litera-ture.
Schistosoma mansoni is a parasitic worm that cause schis-tosomiasis, a serious disease affecting more than 200 mil-lion people in the tropics. Complex multifucosylated oligo-saccharides displayed by glycoprotein and glycolipid sub-sets of larval, egg and adult stages of Schistosoma arethought to play an important role in the immunology ofschistosomiasis. Overkleeft et al. therefore synthesized aspacer-linked GlcNAc monosaccharide and spacer-linkedoligosaccharides based on GlcNAc: α-Fuc-(1�3)-GlcNAc,α-Fuc-(1�2)-α-Fuc-(1�3)-GlcNAc and α-Fuc-(1�2)-α-Fuc-(1�2)-α-Fuc-(1�3)-GlcNAc. This series of linear oli-gosaccharides was used to screen a library of anti-schisto-some monoclonal antibodies by surface plasmon resonancespectroscopy.[198] Other papers describing the preparationof early-stage specific carbohydrate antigens in efforts todevelope good diagnostical tools for detecting schistosomi-asis infection in humans have appeared in the literature.[199]
Takeda et al. performed a detailed study on the synthesisof glycosphingolipids present in the extracellular matrix ofthe cestode Echinococcus multiocularis, possibly involved inthe host–parasite interaction. The linear and branchedglicosphingolipids β-d-Gal-(1�6)-β-d-Gal-1-Cer, α-l-Fuc-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
(1�3)-β-d-Gal-(1�6)-β-d-Gal-1-Cer, β-d-Gal-(1�6)-β-d-Gal-(1�6)-β-d-Gal-1-Cer and β-d-Gal-(1�6)-[α-l-Fuc-(1�3)]-β-d-Gal-(1�6)-β-d-Gal-1-Cer were successfullysynthesized.[200] Subsequently, the carbohydrate cores β-d-Gal-(1�3)-[β-d-GlcNAc-(1�6)]-α-d-GalNAc1-OR, β-d-Gal-(1�4)-β-d-GlcNAc-(1�6)-[β-d-Gal-(1�4)]-α-d-Gal-NAc-1-OR, β-d-Gal-(1�4)-β-d-Gal-(1�3)-β-d-GlcNAc-(1�6)-[β-d-Gal-(1�4)-]-α-d-GalNAc-1-OR and α-d-Gal-(1�4)-β-d-Gal-(1�4)-β-d-GlcNAc-(1�6)-[β-d-Gal-(1�4)]-α-d-GalNAc-1-OR were synthesized by block synthesis.[201]
Finally, Bundle et al. prepared a trisaccharide – β-d-Tyv(1�3)-β-d-GalNAc-(1�4)-β-d-GlcNAc (Tyv =tyvelose, 3,6-dideoxy-d-arabino-hexose) – capping the an-tennary structures of the glycoproteins secreted by Trichi-nella spiralis. These unique glycan chains are thought toplay an important role in the pathogenesis caused by thisnematode.[202]
6. Fungal Infections
The dramatic increase in fungal diseases in recent yearscan be attributed to the improved aggressiveness of medicaltherapy and other human activities. Immunosuppressedpatients, as a population at risk of contracting fungal dis-eases in healthcare settings and from natural environments,are especially affected. In addition, increased prescribing ofantifungals has led to the emergence of resistant fungi, re-sulting in treatment challenges.
Different carbohydrate structures have been identified asantigens capable of eliciting protective immunity againstfungal disease. The mechanisms of immunity vary from an-tibody-mediated immune responses to cell-mediated re-sponses, and even a combination of both. In this context,it is worth mentioning β-glucans as important activators ofthe two arms of the immune system. β-Glucans are β-1,3-glucose polymers branched either in 1,4 or 1,6 fashion.They are able to bind dectin-1 on APCs. Dectin-1 is a PRR,activation of which promotes microbial uptake and phago-cytosis, but in synergy with TLRs it also mediates the pro-duction of cytokines such as IL-12 and TNFα, leading ulti-mately to the initiation of the adaptive immune response.For this reason, β-glucans – or their fragments – can beregarded as a possible class of adjuvants to increase im-mune response to fungal pathogens.[203]
In the planning of vaccines against fungal disease, arange of theoretical questions relating to the safety and effi-cacy of fungal vaccines has been raised, including whethersuch vaccines are necessary, whether they can be made effi-cacious in immunodeficient hosts, whether (in the case ofvaccines against C. albicans) they can prevent disseminateddisease without affecting C. albicans as a member of thenormal microbiota, and whether fungal vaccines againstagents commonly encountered by humans will result in, orpossibly prevent, allergic manifestations.[204] Of course,these questions will be answered as research into fungalvaccines is developed and clinical trials on these prevent-ative agents are initiated.
The yeast-like fungi of the genus Candida are opportu-nistic pathogenic microorganisms capable of causing severeinfections in immunocompromised patients.[205] Thenumber of cases of systemic candidiasis has become amajor medical problem in hospitals, where C. albicans isnow responsible for up to 25 % of nosocomial infections.Treatment of these infections is becoming increasingly diffi-cult, due to improved drug resistance against known anti-fungal compounds.
The main surface antigen of Candida is mannan, a β-(1�2) mannopyranoside polymer that represents the carbo-hydrate part of the cell wall mannoprotein. Although a fewworks about the synthesis of the Candida guilliermondii cellwall have been reported,[206] most research has been di-rected towards the preparation of phosphomannan frag-ments from Candida albicans, the most common etiologicagent in candidiasis. It has been shown that monoclonalantibodies raised against a portion of the C. albicans phos-phomannan in rats were protective against subsequent in-fection,[207] and further studies indicated the active epitopeas a portion of the β-1,2-mannan polymer found in thephosphomannan antigen.
It must be emphasized that the β-O-mannopyranosidicbond present in Candida mannan is one of the most difficultglycosidic linkages to introduce in a synthetic oligosaccha-ride, due to the formidable combination of steric and stereo-electronic factors that weigh against the formation of theβ-mannoside in classical glycosidation protocols.[208] Theinstallation of this challenging β-man configuration was ac-complished by Bundle’s group by use of a ulosyl bromidedonor (compound 182a in Scheme 22) followed by iterativeglycosylations of its p-chlorobenzyl analogue 182b. The de-sired manno configuration was achieved by C-2 reductionwith Li-selectride after each glycosylation step (Scheme 22).Moreover, oligosaccharides were obtained either as propylglycosides (183a–187a) or with aminoethanethiol linkers atthe anomeric positions (183b–187b), suitable for BSA con-jugation.[209]
Scheme 22. Synthesis of the Candida mannans 183–187. DtBMP =2,6-di-tert-butyl-4-methylpyridine.
L. Morelli, L. Poletti, L. LayMICROREVIEWNMR analysis of the unconjugated oligosaccharides
183a–187a, combined with molecular dynamics calcula-tions, revealed the helical characters of these glycan chains,the repeating unit being approximately three residues long.The distinctive conformation of the β-1,2-linked pyranom-annans correlates with the unique immunochemistry ofthese oligomers. A competitive ELISA essay was performedby measuring the affinities of IgM and IgG monoclonalantibodies, generated by immunization of mice with liposo-mal extracts of the C. albicans cell wall, for the propyl gly-cosides 183a–187a. The results showed surprisingly high af-finities for the di- and trisaccharides 183a and 184a in rela-tion to the longer oligomers 185a–187a. This suggested thatprotective monoclonal antibodies recognize short oligosac-charide sequences or the terminal hexose residues of largeroligomers, which could have important implications for thedesign of anti-Candida conjugated vaccines.[210]
In order to verify this hypothesis, a new synthetic path-way leading to gram-scale preparation of mannose oligo-mers was developed by Bundle et al. for the synthesis ofthe tetrasaccharide 188 (Scheme 23). The manno oligomers183b, 184b and 188 were conjugated to BSA or TT proteinthrough adipyl linkers.[211] When the three BSA-glycoconju-gates were titred with the protective monoclonal antibodiesIgG C3.1 and IgM B6.1 on ELISA plates, all three antigensstrongly bound both antibodies, which showed no prefer-ence for any of the three antigens even at high antibodydilutions, at which fine specificity effects might become evi-dent. Their almost identical binding curves strongly sug-gested that the binding sites of both antibodies are largelydirected toward recognition of the common terminal disac-charide present in the three antigens, implying that a disac-charide or trisaccharide glycoconjugate would be an excel-lent candidate for a commercially viable synthetic conjugatevaccine.
Scheme 23. Conjugation of the Candida mannose oligosaccharides183b, 184b and 188 to the carrier proteins TT and BSA.
The same group subsequently investigated clustering ofoligosaccharide motifs in the search for an efficient andmultiple presentation of the C. albicans β-mannan antigenicdeterminant.[212]
To this end, the glucose-derived glycoside 190(Scheme 24), bearing an azide group at the glycosidic linkeras latent amine for eventual conjugation to a protein, waschosen as the multivalent core. The dimannose antigen181[211] was coupled to the multivalent core, and the re-sulting compound was conjugated to BSA and TT proteinsthrough adipyl linkers (�191a–c, Scheme 24). Unfortu-nately, the degrees of incorporation of antigen moieties onboth proteins were low (MALDI-TOF analysis) and com-parable to the loadings of non-clustered mannose disaccha-rides.
Scheme 24. Synthesis of the clustered glycoconjugates 191a–c.
The clustered glycoconjugates 191 were used to vaccinatemice and rabbits, and the sera were titred against the un-clustered compounds 183b-BSA, 184b-BSA, 183b-TT and184b-TT. The clustered glycoconjugates 191a and 191c werehighly immunogenic in rabbits but significantly lessimmunogenic in mice. However, the analysed sera unexpec-tedly exhibited titres approximately 10 times lower than thenative polysaccharide cell wall. Moreover, sera from rabbitsimmunized with the more easily prepared disaccharide andtrisaccharide conjugates 183b-TT and 184b-TT elicitedhigher antibody levels against synthetic and native antigen.
Because the conjugate of the trisaccharide epitope is sim-pler to prepare and yields sera with high titres for the im-munizing epitope and especially for the cell wall β-mannan,a glycoconjugate vaccine based on 184b was subsequentlydeveloped. The concept behind this new-generation vaccinewas the preparation of two- or three-component constructs(compounds 192a–b, Figure 31) bearing the carbohydrate
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Figure 31. Two- and three-component Candida vaccines.
antigen, a Th cell peptide epitope (peptide p458m from themurine 60 kDa heat shock protein) and (for construct 192b)an immunostimulatory peptide epitope from human inter-leukine-1β (IL-1β).[213] Sera from mice immunized withcompounds 192a and 192b were analysed by ELISA withMan3-specific antibodies and displayed high antibody titresagainst (Man)3-BSA conjugated antigen and, most import-antly, against a crude extract of C. albicans cell wall. Inter-estingly, immunization with the three-component vaccine192b incorporating the peptidic IL-1β adjuvant epitope re-producibly leads to higher carbohydrate-specific antibodylevels.
Finally, β-(Man)3-based two-component vaccines weresynthesized by conjugating the trisaccharide 184b to sixfungal cell wall peptides found in C. albicans cell wall pro-teins, selected by algorithm peptide epitope search.[214]
These were to serve as carriers, hopefully providing an ad-ditional protective epitope, inducing protection against fun-gal strains not producing the carbohydrate epitope. ELISAtesting of the six glycoconjugates demonstrated their immu-nogenicity in BALB/c mice, with production of specific an-tibodies against both the glycan epitope and the carrierpeptide. This work demonstrated the feasibility of generat-ing a synthetic glycopeptide-based vaccine in which peptideand saccharide components are small and chemically welldefined (see also Section 7). In addition, this approach hasthe advantage that the peptide carrier is derived from theinfectious agent of interest, offering the possibility of a dualprotective immune response against both the glycan epitopeand the peptide carrier.
Further studies by Bundle and co-workers focused on theantigenic properties of thio analogues of β-Man oligosac-charides.[209b,215] In particular, the thio-trisaccharides 195and 198 (Scheme 25) were prepared by a synthetic routethat alternatively exploited the already applied C-2 oxi-dation/reduction sequence to achieve the β-manno configu-ration after glycosylation of glucose donors and a SN2 reac-tion with thioacetate to introduce the sulfur atom at the C-2 position in the manno configuration.[216] ELISA testingof compounds 195 and 198 revealed a higher immunogenicactivity for the trisaccharide 198 while also confirming theclose conformational similarity of the sulfur-containing oli-gosaccharides to native O-linked oligosaccharides.
Scheme 25. Synthesis of thio analogues of Candida mannans. TCA= trichloroacetimidate.
More recently, the activity of Bundle’s group has beendirected towards the preparation of monodeoxy and mono-O-methyl analogues of the β-Man disaccharide epitope, inorder to gain deeper knowledge of the recognition elementsof the protective epitope.[217]
6.2. Infection by Cryptococcus spp.
Cryptococcus neoformans is a fungus that is the causativeagent of cryptococcosis, a disease that primarily affects in-dividuals with impaired immune systems, such as those withadvanced HIV infection or the recipients of organ trans-plants and immunosuppressive therapies.[218] C. neoformansis unusual among eukaryotic pathogens in having a CPScomposed primarily of glucuronoxylomannan (GXM), rec-ognized as a critical attribute for pathogenicity.[219]
L. Morelli, L. Poletti, L. LayMICROREVIEWC. neoformans is divided into serotypes A–D, depending
on the structure of the CPS. All serotypes have a commonlinear α-1,3-linked mannosyl backbone with β-glucopyr-anosyluronic acid and β-xylopyranosyl substituents (Fig-ure 32).[220] In addition, the backbone is substituted withvariable amounts of 6-O-acetyl groups, with serotype D be-ing the most heavily O-acetylated and serotype C the leastheavily O-acetylated.[221]
Figure 32. Model structures of GXM of C. neoformans sero-types A–D.
Numerous studies have established that antibodies toGXM are protective in mouse models of infection. Unfor-tunately, cryptococcal infection either elicits low levels ofcapsule binding antibodies and/or the antibodies producedare nonprotective, so the contribution of the natural anti-body response to host defence is uncertain.[222] However,conjugation of C. neoformans CPS to proteins results inhighly immunogenic compounds that can elicit high-titreantibody response.
The synthesis of the repeating units of the polysaccharidefrom C. neoformans serotypes A–D is hampered by the rela-tively poor reactivity of the 2-axial hydroxy group of mann-ose and the steric hindrance caused by the 2,3-substitutionof Man residues.
The synthesis of trisaccharide and tetrasaccharide frag-ments corresponding to structures in capsular polysaccha-rides of C. neoformans was reported in the 1990s.[223] Morerecently, Kong et al. reported the preparation of serovar A,B and C oligosaccharides in the shifted sequence shownin Figure 33. All approaches followed a blockwise strategyincluding the glycosylation with the GlcA moiety at the endof the synthesis, due to its difficult manipulation both underbasic and under acidic conditions. Whereas the preparationof the serotype A hexasaccharide 200 was obtained througha limited number of protection steps,[224] the synthesis of201 (serotype B) required a more detailed study, due to thesterically hindered introduction of the GlcA unit at the O-
2 position of the reducing mannose.[225] Finally, the sameauthors reported the synthesis of the heptasaccharide frag-ment of O-deacetylated GXM of C. neoformans serotype C202, by a [3 + 2 + 2] glycosylation sequence.[226]
Figure 33. Synthetic repeating units of C. neoformans CPS serov-ars A, B and C.
Oscarson et al. followed a similar block strategy in ap-proaching the synthesis of the C. neoformans CPS back-bone. Through the use of thioethyl glycosides as glycosyldonors, they prepared the xylose-containing building blocks204–206 (Scheme 26), together with the acceptor 203. Thesewere assembled to provide the penta- and hexasaccharides
Scheme 26. Synthesis of xylose-containing C. neoformans CPS frag-ments.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
207a–c, in which building blocks containing glucuronic acidcan be introduced to produce structures corresponding toCryptococcus serotypes A–C.[227]
After these preliminary studies, the preparation of frag-ments containing glucuronic acid was started. However, theformation of the uronic-acid-containing building blockswas complicated by the (possible) presence of acetategroups in the target compounds. This made the use of thisester as protecting group unfeasible and required an orthog-onal protection for the carboxyl moiety. Better yields wereobtained by oxidation of a glucose moiety at C-6 after theglycosylation step (compound 209, Scheme 27). In this waythe glucuronic-acid-containing pentasaccharide 210 was ob-tained.[228] This structure had previously been synthesizedby other workers,[229] but their pathway did not include thepossibility of elongation or the presence of acetyl groups orconjugation of the unprotected target structures.
Scheme 27. Synthesis of C. neoformans CPS fragments containingglucuronic acid. TBDMS = tert-butyldiphenylsilyl; TBAF = tetra-butylammonium fluoride; TEMPO: 2,2,6,6-tetramethyl-1-piperid-inyloxy.
The pentasaccharide 207b and the disaccharide 211 werecoupled to provide a heptasaccharide (Scheme 28) which,once deprotected, represented the major structural motif ofthe most common clinical isolate of C. neoformans GXM(compound 212). This compound was coupled to HSA(squarate methodology, compound 213) and to biotin (com-pound 214). The glycoconjugate compound 213 proved tobe immunogenic, eliciting high-titre IgG responses in micewhen given with complete Freund’s adjuvant.[230]
Further immunological properties of this heptasacchar-ide were investigated in vivo. Unfortunately, monoclonalantibodies (mAbs) generated from mice immunized withthe glycoconjugate compound 213 produced the character-istic punctuate immunofluorescence associated with non-protective mAbs.
This compound is therefore not a suitable vaccine candi-date and the approach of making a synthetic oligosaccha-ride vaccine that elicits a protective immune response re-mains to be proven for C. neoformans, possibly throughlarger oligosaccharide chains.[231]
Scheme 28. Synthesis of conjugated C. neoformans CPS fragments.
7. Anticancer Vaccines
The stimulation of the patient’s immune system to eradi-cate cancer through the use of a vaccine construct that canelicit a specific immune response against the cancer is theconceptual basis of cancer immunotherapy. Obviously, thisis a very attractive prospect, because of its potential as ahighly powerful and specific approach for cancer cure.However, whereas classical viral or bacterial vaccines aregenerally employed prophylactically to prevent future infec-tious diseases, cancer vaccines are conceived as a thera-peutic approach to induce an immune response capable oftargeting an already existing disease. More specifically, themost recent vaccine strategies aim to provide enhanced pro-tection against tumour reoccurrence and metastasis oncethe tumour has been removed or reduced by surgery, radia-tion or chemotherapeutic treatment.
Whereas previous approaches were based on the passiveadministration of antitumour monoclonal antibodies (pass-ive immunotherapy), active tumour immunotherapy isbased on the theory that tumours possess specific antigensthat can be recognized when presented to or processed byan appropriately trained immune system. In particular, ontheir surfaces tumour cells express Tumour-AssociatedCarbohydrate Antigens (TACAs), typically displayed aseither glycoproteins or glycosphingolipids, that are exclu-sively expressed by tumours and not by normal tissues, orare expressed by normal tissues in a quantitatively andqualitatively different form.[232] Most of the human cancers,for example, are characterized by aberrant glycosylation: tu-mour cells might over-express truncated versions of oligo-saccharides, unusual terminal oligosaccharide sequences,
L. Morelli, L. Poletti, L. LayMICROREVIEWand increased sialylation of cell-surface glycolipids and gly-coproteins.[233] These abnormal glycosylation patterns playa key role in the induction of invasion and metastasis, andare closely correlated with the survival rates of cancerpatients. As a consequence, these antigens provide viabletargets for the development of tumour-specific carbo-hydrate-based vaccines. However, the isolation of adequatequantities of carbohydrate antigens in high purity and withstructural integrity from natural sources is an extremely dif-ficult task. This is the reason why vaccines based on syn-thetic TACAs are emerging as a promising therapy for in-ducing the human immune system to generate tumour-spe-cific immune responses.
A number of practical issues have to be addressed for thedevelopment of an effective and useful vaccine of this type.Firstly, TACAs are usually poorly immunogenic and induceT-cell-independent immune responses, whereas T-cell-medi-ated immunity is crucial for cancer immunotherapy. As de-tailed in previous sections, covalent coupling of carbo-hydrates to immunogenic protein carriers converts theminto T-cell-dependent antigens, leading to a class switchfrom low-affinity IgM antibodies to high-affinity IgG anti-bodies. A second important issue is that TACAs are self-antigens, because they can be expressed, albeit in low con-centrations, in normal tissues or have strict structural simi-larity with normal antigens. Consequently, most TACAs aretolerated by the patient’s immune system and escape theimmune defence mechanisms. In addition, several differentTACAs can be associated with any given cancer type. Thelevels and types of cell surface carbohydrates can vary overthe lifetime of a single tumour cell, and this aspect must betaken into consideration in designing a vaccine destined forclinical use.
Figure 34. Structures of tumour-associated gangliosides.
In summary, there is no doubt that the development ofa clinically useful carbohydrate-based anticancer vaccine isa very challenging task, but it would be widely justified byits enormous potential therapeutic benefits and tremendousimpact on public health.
7.1. Brief Description of TACAs
Tumour-associated carbohydrates have generally beenclassified into four groups.
Gangliosides, such as GM2, GM3, GD2, GD3 and fuco-syl-GM1 (Figure 34), are linked to lipids as glycosphingoli-pids and detectable on normal cells but are overexpressed invarious cancer types (melanoma, glioma, lung cancer, coloncancer, renal cancer and prostate cancer).
Blood group determinants are exposed as glycosphingoli-pids identified as adhesion molecules and, consequently,implicated in tumour invasion and metastasis. The most im-portant blood group determinants associated with humantumours comprise the Lewis antigens: namely sialyl Lewisx
(SLex, 220), SLea (221), Ley (222) and KH-1 antigen (223),consisting of the heterodimeric Ley-Lex heptasaccharide(Figure 35).
Ley, for example, is expressed in colon and liver cancer,but is also implicated in breast, prostate and ovarian tu-mours. The KH-1 antigen is involved in a variety of humantumours, such as colon adenocarcinoma, but has neverbeen isolated from normal colonic tissue, thus representinga highly specific tumour marker.
Glycophorins are typically associated to epithelial cellmucins. Mucins are a family of densely glycosylated high-molecular-weight proteins implicated in a variety of epithe-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Figure 35. Structures of tumour-associated blood group determinants.
Figure 36. Structures of selected tumour-associated antigens from glycophorins and the globo series.
lial cancers, such as breast, prostate and ovarian cancers.Glycophorins are α-O-glycosylated to serine/threonine resi-dues in clustered form in mucin repeats and include Tn,sialyl-Tn (STn) and the Thomsen–Friedenreich (TF) anti-gens 224–226, respectively (Figure 36). Interestingly, theseantigens are overexpressed in many carcinomas but they arelittle exposed in normal tissue. STn and related Tn and TFantigens are therefore very attractive targets for cancer vac-cine development.
The globo series includes the Globo H hexasaccharide227, isolated as a glycolipid from human breast cancer cellsthrough the use of a monoclonal antibody MBr-1, and theglobotriaosyl ceramide Gb3 (228, Figure 36). Globo H isadditionally expressed in colon, lung, ovary, prostate andsmall-cell lung cancers.
7.2. Glycoconjugate Cancer Vaccines
Because of the weak immunogenicities of TACAs, thedesign of carbohydrate-based cancer vaccines initially fol-
lowed the same successful approach used for bacterialcarbohydrate antigens, involving the conjugation of saccha-ride antigens to carrier proteins. A number of different car-rier proteins have been investigated in this regard (vide in-fra), leading to T-cell-dependent glycoconjugates. In ad-dition, anticancer vaccine candidates are often administeredin combination with an immunoadjuvant to provide en-hanced stimulation of the innate immune response. Finally,the conjugation chemistry and the nature of the linker em-ployed to attach the carbohydrate antigen to the carrierprotein are further crucial issues to take into considerationin the design of cancer vaccines. Appropriate choice of allthese elements (i.e., carrier protein, adjuvant, linker andconjugation method) plays a fundamental role in determin-ing the clinical efficacy of a glycoconjugate cancer vaccine.
First-generation glycoconjugate vaccines were developedby Danishefsky’s group in the mid-1990s and were mono-valent in nature, each containing a single type of TACAconjugated to an immunogenic carrier molecule, typicallyKLH. Most of the monovalent constructs reported in the
L. Morelli, L. Poletti, L. LayMICROREVIEWliterature were assembled with use of the heterobifunctionallinker 4-(4-maleimido-methyl)cyclohexane-1-carboxylicacid hydrazide (MMCCH, Figure 37)[234] for conjugation toKLH.
Figure 37. Monovalent anticancer vaccines based on the MMCCHlinker.
Moreover, to improve immunogenicity, these vaccinesmust be co-administered with an immunoadjuvant (QS21or Detox).
One of the most promising mono-epitopic constructs wasthe Globo H-KLH conjugate, assembled with fully syn-thetic Globo H hexasaccharide 227 (Figure 36).[235] The to-tal synthesis of the Globo H antigen provided an excellentvalidation of the glycal assembly strategy developed byDanishefsky and co-workers.[236] In combination with theadjuvant QS-21, the Globo H-KLH conjugate showedhigh-titre IgM and also (weaker) IgG responses.[237] Morerecently, an improved and more efficient synthesis of theGlobo H hexasaccharide was reported.[238] The new synthe-sis was required for the scale-up production of Globo H,in order to advance Globo H-based anticancer vaccines toclinical trials.
Other important examples of these monovalent vaccinecandidates include KLH conjugates of the synthetic Ley
pentasaccharide 222 (Figure 35),[239] glycophorin-KLHconjugates,[240] KLH conjugates of the synthetic KH-1 non-asaccharide antigen 223 (Figure 35) allyl glycoside,[241] andthe conjugate of the fucosyl GM1 hexasaccharide 219 (Fig-ure 34).[242] In addition, the synthesis of the KLH conjugateof the sLea hexasaccharide 221 (Figure 35) has very recentlybeen reported.[243] Mice immunized with this conjugate pro-duced high titres of IgM and IgG antibodies that were veryspecific against sLea and did not show any detectable crossreactivity with the structurally related antigens Ley, Lex andsLex.
Most of these mono-epitopic conjugate vaccines reachedclinical trials[244] but failed to elicit sufficient T-cell-medi-ated immunity (IgG antibodies) in cancer patients. How-ever, these monovalent conjugates were useful for demon-strating the feasibility of developing tumour-specific anti-cancer vaccines targeting carbohydrate antigens. These re-sults stimulated the development of synthetic methodolo-gies for the preparation of glycopeptide-based vaccines cap-able of mimicking tumour cell surfaces. In particular, in themucin family of glycoproteins, cell surface carbohydrate ep-itopes are often displayed in clusters of two to five. Becausethese clusters appear to be the preferred targets for mono-clonal antibodies,[245] it was reasoned that the synthesis of
clustered TACAs as glycopeptides might, upon protein car-rier conjugation, afford vaccine constructs of enhanced im-munogenicity. The synthesis of glycopeptides, especiallystereochemical control in the formation of the α-O-glycos-idic linkages between the carbohydrate domains and thekey amino acids serine and threonine, is a greatly challeng-ing task. In this context, Danishefsky and co-workers devel-oped the “cassette” strategy,[246] a generally reliable proto-col characterized by a modular approach in which the basicbuilding block is stereospecifically α-O-linked to a serineor threonine residue. The resulting glycosylamino acid is ageneral block to be employed in subsequent glycosylationswith suitable glycosyl donors for the synthesis of the glyco-peptide. The logic of the cassette approach, in comparisonwith the traditional convergent strategy, is illustrated inScheme 29.
Scheme 29. The logic of the cassette approach. PG = suitable pro-tecting group; R = selectively removable protecting group; R1 = H(serine) or Me (threonine).
Because all members of the glycophorin family containan N-acetylgalactosamine moiety α-O-linked to a serine (orthreonine) amino acid as a highly conserved motif, the
Scheme 30. Synthesis of αGalNAc-based cassettes. R = H (serine)or Me (threonine). TIPS = triisopropylsilyl; TBS = tert-butyldi-methylsilyl; Fmoc = fluoren-9-ylmethoxycarbonyl.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Figure 38. Structures of KLH-conjugated clustered vaccines.
αGalNAc 1-O-Ser(Threo) cassette was synthesized with adifferentiable acceptor site in order to assemble the glyco-phorin antigens (Scheme 30).
In this way, the crucial and challenging α-O-linkage toserine/threonine has been installed on a simple monosac-charide in a early phase of the synthesis, thus avoiding di-rect coupling to a fully elaborated and already complex sac-charide donor.
The cassette strategy was successfully exploited for thepreparation of trimeric clustered glycopeptides containingTn,[247] TF,[246,248] STn,[249] 2,6-sialyl TF[250] and Ley[251] tu-mour antigens, which were conjugated to KLH through theheterobifunctional linker m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS, Figure 38).
Only Tn(c)-KLH and TF(c)-KLH clusters, coadminis-tered with adjuvant QS-21, progressed to phase I clinicaltrials with prostate cancer patients.[245c,248,252] In the ma-jority of patients both conjugates elicited high titres of IgMand IgG antibodies, which were still detectable for anumber of weeks after the treatment but eventually declinedover time. These promising, albeit not conclusive, resultswere further confirmed by the levels of prostate-specific an-tigen (PSA) in the treated patients, which either remainedstable or, in some cases, decreased after the administration.
All the approaches to carbohydrate-based cancer vac-cines described so far were designed to target one antigenper vaccine. However, many cancer types can express sev-eral different TACAs at varying levels during the phases ofcellular development. Consequently, a monovalent con-struct based on a single antigen might neglect a significantpopulation of tumour cells. The simultaneous presentationof several different TACAs to the immune system shouldallow for the induction of a broader and more potentimmunogenic response. In theory, this goal could beachieved by use of a mixture of monovalent constructs,each separately conjugated to the carrier protein. Althoughpreliminary investigations demonstrated that the immuno-genicities of the individual components remain unchangedin polymolecular formulation of such a kind,[253] a signifi-cant drawback of this approach is the need to use an in-creased amount of carrier protein, which can override theimmunogenicity of the antigen itself. The best option foraddressing this issue was the development of unimolecularmultivalent conjugate vaccines, in which each of the compo-
nent antigens is displayed in the framework of a single con-struct that, in turn, is covalently linked to the carrier pro-tein in a single conjugation step.
The first examples of constructs based on this conceptwere the trivalent compounds 238 (Figure 39), consisting ofthe TF, Tn and Ley antigens α-O-linked to serine residues,and 239, in which the Globo H, Ley and Tn carbohydratedomains are anchored to the peptide backbone through anon-natural amino acid called hydroxynorleucine, contain-ing a tetramethylene linker. Both glycopeptides were pre-pared by total synthesis by the cassette strategy and solu-tion-phase peptide synthesis[254] and were conjugated toKLH through the MBS linker.[255] The conjugate 239 wasfound to be more antigenic than 238 in preliminary ELISAinvestigations. Furthermore, the IgM and IgG antibodiesraised in response to 239 reacted strongly with tumour cellsknown to express each tumour-associated antigen selec-tively.[255]
Figure 39. Structures of the multivalent glycopeptides 238–240.
After these preliminary findings, new, highly elaboratedpentavalent and hexavalent constructs containing carbo-hydrate antigens specifically associated with breast andprostate cancers were synthesized and immunologicallyevaluated.[256] These were a pentavalent conjugate vaccine
L. Morelli, L. Poletti, L. LayMICROREVIEWincluding the Globo H, STn, Tn, Ley and TF antigens[256a]
and its hexavalent version containing the GM2 oligosaccha-ride 215 (Figure 34) as an additional carbohydrate epito-pe.[256b] The Ley antigen, unlike the GM2 epitope, wasfound to be unable to induce the generation of specific anti-bodies when presented in the context of the unimolecularmultiantigenic system, so a second pentavalent glycopeptidein which the Ley was replaced with the GM2 antigen wasprepared.[256b] This glycopeptide was efficiently conjugatedto KLH through the MBS linker (construct 240, Figure 39)and subjected to preclinical immunogenic evaluation inmice in the presence of QS-21 as an adjuvant,[256c] produc-ing excellent IgG and IgM antibody titres against each ofthe five carbohydrate antigens. Moreover, the antibodiesraised in the resulting serum are highly reactive to cancercells overexpressing these antigens. Thanks to these promis-ing results, phase I clinical trials with this multiepitopicconjugate vaccine are expected to start shortly.
In the most recent evolution of synthetic glycoconjugateanticancer vaccines, the possibility has been considered thatthe peptide backbone, beyond its role as a linker to carrierprotein, might also provide for additional antigenic markercapability. For this purpose, action of peptide fragments ofthe mucin family not only as B cell epitopes for the pro-duction of antibodies against mucins, but also as T cell epi-topes to stimulate T cell proliferation has been taken intoconsideration. A new kind of anticancer vaccine, featuringboth a carbohydrate-based antigen and a mucin-derived
Scheme 31. Synthesis of the Gb3-MUC5AC thioester cassette 245.
peptide-based marker in an alternating pattern, has thusbeen designed. Such a construct can include either multiplecopies of the same carbohydrate antigen or a combinationof diverse carbohydrate antigens associated with a particu-lar type of cancer, providing access to a new generation ofanticancer vaccines with potentially enhanced efficiency, be-cause they should better mimic the natural tumour cell sur-face.
In the first example of this “dual-acting” vaccine, Gb3(globotriaosyl ceramide) carbohydrate antigen (228, Fig-ure 36), overexpressed on ovarian tumour cell surfaces, andthe tandem repeat of MUC5AC mucin antigen (a sequenceof eight amino acids), used both as a linker and as a T cellepitope, were assembled in a alternating pattern.[257] Thesynthesis of the construct was based on the Gb3-MUC5ACthioester cassette 245, which in turn was derived from theGb3 glycosylamino acid 243.
The synthesis of 243 was accomplished through a cross-metathesis reaction between the Gb3 pentenyl glycoside 241and the non-natural N-Fmoc-protected hydroxynorleucinebenzyl ester 242. The cassette 245 was then completed bycondensation of 243 with the synthetic MUC5AC octapep-tide containing a C-terminal thioester group. Moreover, 243was further coupled with tert-butyl N-(3-aminopropyl)carb-amate to provide 244, containing the linker for conjugationto the protein carrier (Scheme 31). In the next stages of thesynthesis, the thioester cassette 245 was coupled with theglycosylamino acid 244, followed by Fmoc removal and it-
Synthetic Oligosaccharide Antigens for Vaccine Formulation
eration to provide the fully assembled trivalent glycopeptide246. Finally, 246 was conjugated to KLH carrier proteinthrough the MBS linker to afford the construct 247(Scheme 32).
In a very similar approach, the pentavalent glycopeptideof 240 (Figure 39) was covalently linked to a tandem repeatof unglycosylated human tumour-associated mucin MUC1.The resulting construct possesses a linker suitable for pro-tein carrier conjugation.[258] The results of the immunologi-cal studies performed on these interesting conjugate vac-cines are expected in the near future.
7.3. Fully Synthetic Carbohydrate-Based Cancer Vaccines
Although glycoconjugate cancer vaccines show promis-ing immunological activities, a number of issues have led tocurrent debate on their future development. In Section 3 wehave already mentioned the difficulties involved in controlof the conjugation chemistry, which can affect the reprodu-cibility of the immune response. Additionally, protein carri-ers are highly immunogenic and can elicit strong B cell re-sponses, which can lead to the attenuation, or even the sup-pression, of the antibody response against the carbohydrateepitope. Lastly, linkers that are employed for the conjuga-tion of carbohydrates to proteins can also be immunogenic,giving rise to interference with the immune response. As aconsequence, the next stage in the development of antican-cer vaccines was the design of new types of constructs de-void of any unnecessary immunogenic components, con-taining only the elements prerequisite for eliciting a full (in-nate and adaptive) immune response. The first incarnationof this concept were fully synthetic two-component vac-cines, in which TACAs (a B cell epitope) are covalentlylinked to a TLR ligand (the adjuvant), which activatesAPCs, thus enhancing the adaptive immune response speci-fically directed against the carbohydrate antigen. Inspiredby the seminal investigation of Toyokuni et al.,[259] Dani-shefsky and co-workers conjugated monomeric Ley and tri-meric Ley(c),[260] trimeric Tn(c),[252] and the pentavalent gly-copeptide 240[256a] to the immunogenic lipopeptide Pam3-Cys-Serine. When co-administered to mice with the immu-noadjuvant QS-21, these vaccines elicited high titres of anti-bodies that recognized the natural epitope, but mainly ofthe IgM type, without inducing a class switch to IgG anti-bodies.
The preparation of an anticancer vaccine candidatebased on the calix[4]arene scaffold carrying four copies ofa (thio)glycomimetic of the Tn antigen (with sulfur replac-ing the anomeric oxygen) and the immunoadjuvant Pam3-CysSer has recently been reported.[261] The antigenicity ofthe tetravalent calixarene vaccine was compared with thatof a monovalent construct, and displayed a fourfold in-crease in IgG antibody titres due to the multivalency intro-duced by the calixarene scaffold.
A number of recent reports describe a new group ofstructurally distinct bacterial polysaccharides that behavelike traditional T-cell-dependent antigens: they can directly
activate CD4+ T cells both in vivo and in vitro through theMHC-II-dependent pathway without protein conjuga-tion.[262] These molecules have the common characteristicof presenting a zwitterionic charge motif distributed alongthe chain, (i.e., they contain both positive and negativecharge centres within a repeating unit structure; zwitter-ionic polysaccharides, ZPSs).
Andreana and co-workers reported the first example ofan entirely carbohydrate vaccine candidate containing theTn antigen chemically conjugated to PSA1, one of the cap-sular polysaccharides of the commensal bacterium Bacte-roides fragilis consisting of zwitterionic tetrasaccharide re-peating units (Figure 40).[263]
Figure 40. Structure of the Tn-PSA1 conjugate 248.
The immunization studies revealed that the Tn-PSA1conjugate 248 elicited exceptionally high titres of specificantibodies, even in the absence of an external adjuvant.This result could be explained by the assumption thatPSA1, and also other naturally occurring ZPSs, would actas a TLR-2 agonist, as recently suggested.[264]
In quest for more efficient anticancer vaccines based onTACAs linked to specific peptide sequences as helper T cellepitopes, Kunz and co-workers designed and synthesizedtwo-component vaccines consisting of tumour-associatedMUC1 glycopeptides conjugated to a number of helperT cell epitopes. The tumour-associated mucin MUC1 isstrongly overexpressed on epithelial tumour cells. Becauseof misregulation of certain key glycosyltransferases, the gly-can pattern of tumoural MUC1 is distinctly altered relativeto that of a normal cell and is characterized by the presenceof truncated and prematurely sialylated saccharide antigenssuch as the Tn, TF and various sialylated antigens.[265] As aresult, the average saccharide length of MUC1 on epithelialtumour cells is much shorter than that on normal epithelialcells, making the peptide backbone more easily accessibleto the immune system. Both the saccharide and the peptidestructure therefore contribute to the tumour-associated epi-topes.
Various MUC1-derived glycopeptides, composed of STn,(2,6)-STF, (2,3)-STF and (2,3–2,6-disialyl)-TF antigenslinked through threonine to the complete (20 amino acids)MUC1 tandem repeat, were synthesized by solid-phase
L. Morelli, L. Poletti, L. LayMICROREVIEWtechniques. Moreover, the STn-containing MUC1-glyco-dodecapeptide was conjugated to BSA protein[266] andto a helper T cell peptide epitope from ovalbumin(OVA323–339),[267] which showed that both constructs areable to induce specific antibodies against MUC1 glyco-peptides. In a subsequent study, the same authors describedthe synthesis of vaccine constructs containing mono- (249,Figure 41), di- (250) and triglycosylated (251) complete tan-dem repeat peptides linked through nonimmunogenicspacer amino acids to the OVA323–339 T cell epitope.[268]
Figure 41. Vaccine constructs based on MUC1-derived glycopep-tides.
The STn antigen was incorporated in all three vaccines,whereas the di- and triglycosylated peptides contained oneand two additional Tn antigens, respectively. Transgenicmice were immunized with the synthetic conjugates. Onlythe mono- and diglycosylated vaccines 249 and 250 dis-played high antibody titres of the IgG type, all specific tothe MUC1-glycopeptide antigens, showing that both thesaccharide antigen and the peptide backbone are importantfor the epitope recognition. An even more efficient con-struct was synthesized by conjugation of the STn-MUC1glycopeptide antigen to TT as a carrier protein through thesquarate linker (vaccine 252, Figure 41)[269] The immuniza-tion studies showed that the STn-TT vaccine 252 inducesan extremely selective and exceptionally strong immune re-sponse, much stronger than the corresponding monoglycos-ylated OVA323–339 conjugate 249.
Very recently, similar results were obtained in immuniza-tion studies performed with a similar synthetic TF-MUC1glycopeptide conjugated to TT protein. In addition, aclosely related conjugate containing an analogue of the TFantigen 226 with fluorine substituents at the 6- and 6�-posi-tions of the disaccharide [(F)2TF] was also investigated.[270]
Both conjugates elicited strong and specific immune re-sponses and induced antibodies predominantly of the IgGtype. Notably, the antibodies induced by the (F)2TF-MUC1conjugate were found to cross-react with the natural TF-MUC1-BSA conjugate and to bind strongly to the epithe-lial tumour cells of the MCF-7 mammary adenocarcinomacell line, indicating that the primary OH groups of the TFantigen can be replaced by fluorine without reducing theimmunogenicity of the vaccine.
To address the issue of possible carbohydrate epitopesuppression by the TT carrier protein, a different form ofvaccine was recently synthesized. Here, the MUC1 glyco-peptide (monoglycosylated variously with Tn, or with TF,or with 2,6-sialyl TF tumour antigens) is covalently linkedthrough an oligoethylene glycol spacer to an immunostimu-lating TLR ligand such as the lipopeptide Pam3CysSer-(Lys)4 (Pam3CSK4, Figure 42).[271]
Figure 42. Vaccine constructs composed of monoglycosylatedMUC1 glycopeptide and TLR ligand.
Immunization studies showed that the vaccine elicited astrong and specific immune response, but the antibodytitres were not as high as those for the correspondingOVA[268] or TT[269] conjugates.
The heterogeneity in glycosylation of MUC1-derivedpeptides was targeted with the synthesis and biologicalevaluation of a multiantigenic glycopeptide construct con-taining the universal helper T cell epitope PADRE (the PanHLA DR-binding epitope) and three MUC1 tandem re-peats, two of them carrying the Tn and the TF carbohydrateantigens.[272] IgG antibodies were induced against bothB cell epitopes, and the antisera recognized native tumourepitopes expressed by human MCF-7 cell line.
Another approach to targeting carbohydrate epitopeclustering on tumour cells surface and circumventing thelimitations of glycoconjugate vaccines has been the designof a dendrimeric multiantigenic glycopeptide (MAG) con-taining multiple copies of the Tn antigen together with ahelper T cell epitope. In particular, the MAG was based ona nonimmunogenic four-arm tris-lysine core, each arm ex-tended by a T cell peptide epitope derived from polio virustype 1 (PV)[273] or the PADRE epitope or even a TT-derivedpeptide,[274] and a trimeric Tn antigen linked to a tris-threo-nine tripeptide (Figure 43).
Figure 43. Outline of a MAG-based dendrimeric vaccine construct.
These MAG constructs were examined in mice and non-human primates and found to induce strong anti-Tn IgGantibodies capable of specifically recognizing Tn-expressinghuman tumour cells.[274]
Synthetic Oligosaccharide Antigens for Vaccine Formulation
This study emphasized the increasingly accepted conceptthat both the clustering of the carbohydrate antigens andthe way in which they are presented to the immune systemare crucial parameters in stimulating efficient anti-TACAimmune responses. Moreover, nonimmunogenic molecularscaffolds ensure focusing of the immune response on thesaccharide moiety, decreasing the likelihood of carrier-in-duced epitope suppression.
The use of RAFT as a peptide-based scaffold[118] pro-vides further support for these concepts. The opposite facesof the RAFT can be selectively functionalized with differentantigens by classical ligation chemistry on the side chainsof the lysine (K) residues. A RAFT-based cancer vaccinecandidate, containing four copies of the Tn antigen and twocopies of a T cell epitope derived from PV, was synthesizedby oxime ligation chemistry (compound 253, Figure 44).[275]
Figure 44. Structure of a RAFT-based cancer vaccine candidate.
The vaccine induced Tn-specific IgG antibodies that rec-ognized native Tn antigens on human cells, showing thatthe nonnatural oxime bond did not interfere with the im-mune response. Importantly, the raised antibodies were po-orly reactive towards the RAFT scaffold, indicating that itis nonoimmunogenic.
More recently, Danishefsky’s group reported on the syn-thesis of a very similar construct based on the tetravalentvariant of the macrocyclopeptide 106 employed for the de-velopment of an HIV vaccine (see Section 4.1.1).[121] Theupper face of the scaffold was decorated either with fourcopies of the Tn antigen (compound 254, Scheme 33) orwith four copies of the STn antigen (compound 255,
Scheme 33. Structures of glycopeptides based on a macrocyclopeptide scaffold.
Scheme 33) by use of the corresponding cassettes preparedfrom l-hydroxynorleucine benzyl ester. Moreover, the hexa-valent variant of the Tn-conjugate, as well as the multianti-genic glycopeptide 256, containing both the Tn and the STnantigens (Scheme 33), were also prepared.[276] Interestingly,it was reasoned that the presence of artificially cross-linkedantigens on the surface of the macrocyclic scaffold, besidespreventing undesired spreading of the glycan units, wouldcontribute to better mimicry of the clustered antigen andso enhance the antibody response. To test this hypothesis,the bivalent compound 257 (Scheme 33), containing two Tnantigen analogues cross-linked through a ring closing me-tathesis (RCM) reaction, was synthesized. The conjugationof these constructs to a protein carrier and the immunologi-cal evaluation of the resulting conjugates are expected inthe near future.
The two-component vaccines so far described either in-duced insufficient titres of high-affinity IgG antibodies, be-cause of the lack of a helper T cell epitope (Pam3Cys orsimilar TLR ligand conjugates), or needed external adju-vants to overcome the inherent poor immunogenicities ofcarbohydrate antigens (glycopeptide or protein conjugates).
An anticancer vaccine capable of evoking a focused andcomplete immune response against carbohydrates should beachievable through a suitable combination of the three es-sential components (Figure 45): 1) the TACA, acting as aB cell epitope, which must be properly presented to the im-mune system, 2) a helper T cell epitope, ensuring a T-cell-dependent response and a class switch to IgG antibodies,and 3) an immunostimulating TLR ligand to ensure suf-ficient engagement of APCs, leading to a strong amplifi-cation of the immune response.
Figure 45. Outline of a three-component anticancer vaccine.
L. Morelli, L. Poletti, L. LayMICROREVIEWThe first approach of this kind was pursued in 2005 with
the design and synthesis of a fully synthetic anticancer vac-cine candidate composed of the Tn antigen, the T cell epi-tope YAF peptide, derived from an outer membrane pro-tein of Neisseria meningitidis, and the built-in lipopeptidePam3Cys.[277] This construct was incorporated into phos-pholipid-based liposomes, which are attractive vaccine de-livery systems, and administered to mice. Although onlylow-to-moderate titres of IgG anti-Tn antibodies were de-tected, the results were considered promising enough forfurther development.
More recently, two additional three-component vaccinecandidates were designed. These consisted of a MUC1-Tnglycopeptide as a B cell epitope, a peptide from PV as aT cell epitope, and two variants of immunoadjuvants:Pam2CysSK4 (activator of TLR2 and TLR6) in construct259 (Figure 46) or Pam3CysSK4, an agonist of TLR1 andTLR2, in construct 260.[278]
Figure 46. Outline of the three-component vaccines designed inBoons’ laboratory.
The vaccine 259 was prepared by classical solid-phasepeptide synthesis, the vaccine 260 by liposome-mediatednative chemical ligation (NCL).[279] This method was foundto improve both the rates and the yields of the NCL reac-tions,[280] especially when very hydrophobic reactants wereused. Both vaccine candidates were incorporated into lipo-somes and their immunogenicities were evaluated in murinemodels. The construct 260 (but not 259) elicited exception-ally high titres of IgG antibodies, which were shown to bindto MCF-7 tumour cells.[278]
A number of derivatives related to the vaccine 260 weresubsequently prepared in order to investigate the immuno-logical roles of the various components (Figure 47).[281] Theglycolipopeptide 261, differing from 260 in the presence ofan immunosilent lipidated amino acid in place of the TLR2ligand, induced significantly lower titres of antibodies, dem-onstrating that TLR engagement is critical for optimal anti-genic responses. Furthermore, liposomal preparations of261 with Pam3CysSK4 or MPLA – a well-known TLR4agonist – raised IgG antibodies at comparable titres withthose elicited by 260, although with much reduced abilityof recognition for MCF-7 cells, indicating that the covalentattachment of the adjuvant to B and T cell epitopes is alsocrucial for the specificity of the response.
Finally, the need for covalent attachment of all threecomponents of 260 was further highlighted by the behav-iour of the additional compounds 262 (devoid of the T cell
Figure 47. Structures of derivatives related to the vaccine 260.
epitope) and 263 (devoid of the B cell epitope), which dis-played very low antigenicities when co-administered in lipo-somal preparations.[282]
Another strategy for boosting the immune response toTACAs involves targeting the antigens of interest to APCsin order to activate a mechanism of antibody-dependent an-tigen uptake (for an application of this strategy to anti-in-fluenza vaccines see also Section 4.2.1).[153,283] Very re-cently, the first example of a fully synthetic vaccine candi-date based on this concept has been described.[284] On thebasis of previous observations in which human serum wasreported to contain large amounts of naturally occurringanti-rhamnose antibodies, the authors hypothesized thatthe efficiency of a cancer vaccine could be increased by con-jugation of a helper T cell epitope and B cell antigen to anl-rhamnose (Rha) carbohydrate epitope to trigger the sameantibody-mediated antigen uptake mechanism.
Accordingly, they described the synthesis of the unimo-lecular three-component vaccine candidate 264 (Figure 48),consisting of a Rha epitope, the YAF peptide as a T cellepitope, and the Tn antigen as a B cell epitope, and ex-plored its immunological behaviour. Since laboratory micedo not produce anti-Rha antibodies, they were inducedthrough immunization with a Rha-OVA conjugate.
Figure 48. Structure of the vaccine 264 based on the concept ofantigen uptake mediated by anti-Rha antibodies.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
Scheme 34. Synthesis of the four-component vaccine designed in Dumy’s laboratory.
Vaccine challenges showed that the Rha-YAF-Tn conju-gate 264 raised titres of anti-Tn antibodies only slightlyhigher than in the control group and comparable with thoseinduced when only the YAF-Tn conjugate was inoculated.Despite these disappointing results, this pioneering examplecould open new avenues for the development of more effec-tive anticancer vaccines.
An even higher level of sophistication was recently ac-complished by Dumy and co-workers, who reported thefirst example of a fully synthetic, self-adjuvanting four-component anticancer vaccine candidate.[285]
This construct is composed of a cluster of the Tn antigenas a B cell epitope, the PADRE peptide as a T cell epitope,a peptide fragment from ovalbumin (OVA257–264) to act as aCD8+ cytotoxic T lymphocyte (CTL) epitope, and palmiticacid, which serves as a built-in immunoadjuvant, all ofthem displayed on the RAFT cyclopeptide molecular scaf-fold. The carbohydrate functionalization of the cyclodeca-peptide was carried out by the oxime ligation strategy [i.e.,by coupling four copies of aminooxilated α-GalNAc-ONH2
with glyoxoaldehyde functions (obtained by oxidative cleav-age of serines with sodium periodate)] displayed on the up-per surface of RAFT 265 (Scheme 34). Next, the chimericpeptide OVA257–264-PADRE, separately synthesized, wasextended at the N-terminal end with a palmitic acid moietyto confer the self-adjuvanting property, and finally linkedto the lower face of RAFT 266 through a cysteine residue.The resulting construct 267 was administered to mice in anadjuvant-free setting and induced robust Tn-specific IgMand IgG antibodies that recognized MCF-7 cell lines, strongPADRE-specific CD4+ T cell and OVA257–264-specificCD8+ T cell responses, indicating correct antigen pro-cessing and presentation of both Th cell and CTL epitopes.Importantly, vaccination with the glycolipopeptide 267 pro-duced reduction of tumour size in mice and protection ofmice from lethal cancer cell challenge, and significantly in-hibited the growth of pre-established tumours (MO5cells).[286]
Sialic acid cell glycoengineering[1c,287] is a techniquebased on the observation that enzymes can tolerate someforms of structural modification of their substrates, provid-ing opportunities to exploit the sialic acid biosyntheticpathway to produce unnatural sialic acid analogues andtheir corresponding sialoglycoconjugates on cell surfaces byexposing cells to artificial sialic acid precursors. Specifically,N-acetyl-d-mannosamine is a key intermediate in the bio-synthesis of sialic acid. However, the enzymes involved inthis pathway also accept different unnatural N-acyl deriva-tives of d-mannosamine, producing the corresponding N-acyl neuraminic acid derivatives and their unnatural sialog-lycoconjugates. A number of investigators have combinedcell glycoengineering with vaccines composed of unnaturalTACA analogues to develop a novel immunotherapeuticstrategy. Briefly, after vaccination with a synthetic vaccinecontaining TACA analogues, cancer cells are glycoengine-ered to express the TACA analogue on their surfaces. Sub-sequently, the immune system is suitably activated to attackand destroy cancer cells exposing the TACA analogue, thuscircumventing the problems originating from the self-anti-genicity of many TACAs.
The application of cell glycoengineering in cancer vaccin-ology has been explored by many research groups. For amore detailed description of these approaches, the reader isreferred to more specific accounts.[288]
8. Conclusions
There is no doubt that vaccination is the most cost-efficient strategy to fight infectious diseases, which still havea tremendous impact on public health with enormous socialand financial costs, both in industrialized and in developingcountries.
Traditional vaccines, mainly consisting of live attenuatedpathogens, whole inactivated organisms and inactivatedbacterial toxins, can also produce undesirable side effects
L. Morelli, L. Poletti, L. LayMICROREVIEWand safety problems. Several new approaches to vaccine de-velopment have therefore emerged, such as the use of re-combinant protein subunits, synthetic peptides, plasmidDNA and protein-saccharide conjugates as antigenic mate-rial. The key feature of the last of these is that a T-cell-independent carbohydrate antigen is transformed into a T-cell-dependent immunogen when it is covalently linked toan appropriate carrier protein, and it is capable of evokinga strong, durable and specific immune response against thesaccharide itself. In particular, protein conjugates withchemically well-defined synthetic saccharide antigens showgreat promise, as demonstrated by the spectacular successof the synthetic Cuban vaccine Quimi-Hib.[22] However, theinherent poor immunogenicities of carbohydrates still limitthe efficiencies of many glycoconjugate-based vaccines.Typical examples are cancer vaccines, which are intendednot for preventative therapies, but to provide enhanced pro-tection against tumour relapse and metastasis once the tu-mour has been removed. The efficiency of cancer immuno-therapy based on protein conjugates of TACAs is often di-minished because the high immunogenicity of the carrierprotein can override and weaken the immune responseagainst the saccharide antigens.
The extraordinary progress achieved in the field of im-munology in recent years has led to the formulation of moreand more sophisticated vaccines. In particular, a key break-through has been the discovery that the two distinct armsof the immune response, the innate arm and the adaptivearm, are closely interwoven. Whereas the innate immunesystem, based primarily on APC activation and maturation,constitutes the first defence line of the body against foreignmicroorganisms, the adaptive immune response, mediatedby B and T lymphocytes, recognizes pathogens with highaffinity and provides the fine antigenic specificity requiredfor complete elimination of the infective agent and the gen-eration of immunological memory. The more detailed un-derstanding of this complex machinery has led to the designof fully synthetic vaccine candidates that, unlike semi-syn-thetic glycoconjugates, include only those elements strictlyrequired to elicit robust carbohydrate-specific immune re-sponses. These new findings have been mostly applied incancer. Vaccinology research has recognized that a fullycompetent immune response targeting poorly immunogeniccarbohydrate antigens can be achieved by setting in motionall the distinct components of the immune system, in orderto orchestrate a multifaceted activation of the immune de-fence capable of triggering a powerful and selective re-sponse. In this respect, the last generation’s multi-compo-nent vaccines are very promising tools and provide an at-tractive option for the treatment of cancer. In particular,the four-component model recently proposed from Dumy’slaboratory,[285] which incorporates a TACA as a B cell epi-tope, a T cell epitope, an immunoadjuvant, and a peptidefragment to act as a cytotoxic T lymphocyte (CTL) epitope,could open up unprecedented perspectives in the field ofvaccinology. Although the carbohydrate-protein conjugatesare still the leading and best-established vaccine settingsemployed to control infectious diseases, the extension of
fully synthetic three- and four-component constructs to thearea of antibacterial, antifungal and antiparasite vaccinescould open up a new front line in vaccinology. Some rel-evant examples have recently been reported,[39,40,213] butmore and more are expected in the near future. In particu-lar, the search for and development of new and more ef-ficient immunostimulatory compounds will be crucial in thenext years, because alum is the sole adjuvant so far allowedin vaccine settings for human use.
As far as HIV infection is concerned, a very recent find-ing by Bomsel et al. should be mentioned.[289] They evalu-ated the protective efficacy of a vaccine composed of enve-lope subunit gp41 antigens contained in virosomes, whichis active at mucosal sites before primary infection takesplace and blocks the entry of the virus. Their results clearlychallenge the belief that mucosal protection requires signifi-cantly high levels of antibodies with virus-neutralizing ca-pacity in the blood.
This last example shows that many issues are still openand, in spite of the most recent promising results, furtherpreclinical and clinical research is needed to assess the po-tential and limitations of carbohydrate-based vaccine candi-dates suitably designed to target infectious diseases andcancer.
[1] a) A. Varki, Glycobiology 1993, 3, 97–130; b) R. A. Dwek,Chem. Rev. 1996, 96, 683–720; c) C. Bertozzi, L. L. Kiessling,Science 2001, 291, 2357–2364; d) K. Ohtsubo, J. D. Marth, Cell2006, 263, 855–867.
[2] M. Heidelberger, O. T. Avery, J. Exp. Med. 1923, 38, 73–79.[3] J. B. Robbins, R. Austrian, C.-J. Lee, S. C. Rastogi, G. Schiff-
man, J. Henrichsen, P. H. Mäkelä, C. V. Broome, R. R. Fack-lam, R. H. Tiesjema, J. C. Parke Jr, J. Infect. Dis. 1983, 148,1136–1159.
[4] a) S. Segal, A. J. Pollard, Br. Med. Bull. 2004, 72, 65–81 andreferences cited therein; b) Á. González-Fernández, J. Faro, C.Fernandez, Vaccine 2008, 26, 292–300.
[5] J. J. Mond, A. Lees, C. M. Snapper, Annu. Rev. Immunol. 1995,13, 655–692.
[6] O. T. Avery, W. F. Goebel, J. Exp. Med. 1931, 54, 437–447.[7] a) A. A. Lindberg, Vaccine 1999, S28–S36; b) N. Ravenscroft,
C. Jones, Curr. Opin. Drug Discov. Develop. 2000, 3, 222–231.[8] G. Ada, D. Isaacs, Clin. Microbiol. Infect. 2003, 9, 79–85, and
references therein.[9] a) G. B. Lesinski, M. A. Westerink, Curr. Drug Targets Infect.
Disord. 2001, 1, 325–334; b) A. Weintraub, Carbohydr. Res.2003, 338, 2539–2547.
[10] R. D. Astronomo, D. R. Burton, Nature Rev. Drug Discov.2010, 9, 308–324.
[11] a) T. J. Boltje, T. Buskas, G.-J. Boons, Nature Chem. 2009, 1,611–622; b) D. P. Galonic, D. Y. Gin, Nature 2007, 446, 1000–1007.
[12] a) P. H. Seeberger, Carbohydr. Res. 2008, 343, 1889–1896; b)P. H. Seeberger, Chem. Soc. Rev. 2008, 37, 19–28; c) D. B. Werz,B. Castagner, P. H. Seeberger, J. Am. Chem. Soc. 2007, 129,2770–2771; d) P. H. Seeberger, W. C. Haase, Chem. Rev. 2000,100, 4349–4394.
[13] Freund’s adjuvant is an water-in-oil emulsion containing inacti-vated mycobacteria (complete form), or that does not containmycobacteria (incomplete form).
[14] a) J. B. Robbins, R. Schneerson, N. Engl. J. Med. 1990, 323,1415–1416; b) B. D. Gessner, R. A. Adegbola, Vaccine 2008,26, B3–B8; c) C. Jones, Ann. Acad. Bras. Cienc. 2005, 77, 293–324.
[15] E. Miller, D. Salisbury, M. Ramsay, Vaccine 2001, 20, S58–S67.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
[16] J. A. Scott, Vaccine 2007, 25, 2398–2405.[17] a) J. T. Poolman, Expert Rev. Vaccines 2004, 3, 597–604; b) S.
Lockhart, J. G. Hackell, B. Fritzell, Expert Rev. Vaccines 2006,5, 553–564.
[18] V. Pozgay, Curr. Top. Med. Chem. 2008, 8, 126–140.[19] The term hapten refers to a small molecule usually unable to
induce an immune response unless linked to an immunogeniccarrier.
[20] B. Lepenies, J. Yin, P. H. Seeberger, Curr. Opin. Chem. Biol.2010, 14, 404–411, and references therein.
[21] a) V. Pozsgay, Adv. Carbohydr. Chem. Biochem. 2001, 56, 153–199; b) V. Pozsgay, in: Immunobiology of Carbohydrates (Eds.:S. Y. C. Wong, G. Arsequell), Eurekah.com and Kluwer Aca-demic/Plenum Publishers, New York, 2003, pp. 192–273.
[22] a) V. Verez-Bencomo, V. Fernandez-Santana, E. Hardy, M. E.Toledo, M. C. Rodriguez, L. Heynngnezz, A. Rodriguez, A.Baly, L. Herrera, M. Izquierdo, A. Villar, Y. Valdés, K. Cosme,M. L. Deler, M. Montane, E. Garcia, A. Ramos, A. Aguilar,E. Medina, G. Toraño, I. Sosa, I. Hernandez, R. Martinez, A.Muzachio, A. Carmenates, L. Costa, F. Cardoso, C. Campa,M. Diaz, R. Roy, Science 2004, 305, 522–525; b) V. Fernández-Santana, F. Cardoso, A. Rodriguez, T. Carmenate, L. Peña, Y.Valdés, E. Hardy, F. Mawas, L. Heynngnezz, M. C. Rodríguez,I. Figueroa, J. Chang, M. E. Toledo, A. Musacchio, I.Hernández, M. Izquierdo, K. Cosme, R. Roy, V. Verez-Bencomo, Infect. Immun. 2004, 72, 7115–7123.
[23] World Health Organization. Haemophilus influenzae type B(HiB). World Health Organization website [online], http://www.who.int/mediacentre/factsheets/fs294/en/ (accessed onFebruary 21st 2011).
[24] A. V. Nikolaev, I. V. Botvinko, A. Ross, Carbohydr. Res. 2007,342, 297–344.
[25] a) B. Classon, P. J. Garegg, I. Lindh, Carbohydr. Res. 1988, 179,31–35; b) S. Oscarson, Carbohydr. Polym. 2001, 44, 305–311and references cited therein.
[26] A. Fekete, P. Hoogerhout, G. Zomer, J. Kubler-Kielb, R.Schneerson, J. Robbins, V. Pozsgay, Carbohydr. Res. 2006, 341,2037–2048.
[27] M. M. Levine, K. L. Kotloff, E. M. Barry, M. F. Pasetti, M. B.Sztein, Nat. Rev. Microbiol. 2007, 5, 540–553.
[28] A. Phalipon, L. A. Mulard, P. J. Sansonetti, Microbes Infect.2008, 10, 1057–1062.
[29] The biological repeating unit of the O-SP is [�3)-α-d-Rha-(1�3)-α-l-Rha-(1�2)-α-d-Gal-(1�3)-α-d-GlcNAc-(1] and itssynthesis was reported by P. Kovác, K. J. Edgar, J. Org. Chem.1992, 57, 2455–2467. However, for synthetic reasons the au-thors selected the shifted sequence as the repeating block.
[30] a) V. Pozsgay, Angew. Chem. 1998, 110, 145; Angew. Chem. Int.Ed. 1998, 37, 138–142; b) V. Pozsgay, J. Org. Chem. 1998, 63,5983–5999.
[31] V. Posgay, C. Chu, L. Pannell, J. Wolfe, J. B. Robbins, R. Schne-erson, Proc. Natl. Acad. Sci. USA 1999, 96, 5194–5197.
[32] This impressive result should be considered with caution, how-ever. The O–SP–HSA conjugate was obtained by activationwith cyanogen bromide followed by derivatization with adipicacid dihydrazide and condensation with the carboxyl groups ofthe protein (multiple point attachment). The authors admitthat, because of the random activation with CNBr, conjugatesof this kind have varied and unpredictable compositions, andno reliable method to determine their exact structures (in termsof number of intact repeating units) and to foresee their immu-nogenicities is available. On the other hand, synthetic saccha-rides have defined sizes and compositions, allowing the conju-gation to the protein by single-point attachment, and providingstructures that can be characterized much more accurately.
[33] a) V. Pozsgay, B. Coxon, C. P. J. Glaudemans, R. Schneerson,J. B. Robbins, Synlett 2003, 743–767; b) V. Pozsgay, G. Ekborg,S.-G. Sampathkumar, Carbohydr. Res. 2006, 341, 1408–1427.
[34] V. Pozsgay, J. Kubler-Kielb, R. Schneerson, J. B. Robbins, Proc.Natl. Acad. Sci. USA 2007, 104, 14478–14482.
[35] a) L. A. Mulard, C. Costachel, P. J. Sansonetti, J. Carbohydr.Chem. 2000, 19, 849–877; b) C. Costachel, P. J. Sansonetti,L. A. Mulard, J. Carbohydr. Chem. 2000, 19, 1131–1150; c) F.Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002, 13, 2211–2222; d) F. Belot, C. Costachel, K. Wright, A. Phalipon, L. A.Mulard, Tetrahedron Lett. 2002, 43, 8215–8218.
[36] a) F. Belot, K. Wright, C. Costachel, A. Phalipon, L. A. Mul-ard, J. Org. Chem. 2004, 69, 1060–1074; b) K. Wright, C. Guer-reiro, I. Laurent, F. Baleux, L. A. Mulard, Org. Biomol. Chem.2004, 2, 1518–1527; c) F. Belot, C. Guerreiro, F. Baleux, L. A.Mulard, Chem. Eur. J. 2005, 11, 1625–1635.
[37] A. Phalipon, C. Costachel, C. Grandjean, A. Thuizat, C. Guer-reiro, M. Tanguy, F. Nato, B. Vulliez-Le Normand, F. Belot,K. Wright, V. Marcel-Peyre, P. J. Sansonetti, L. A. Mulard, J.Immunol. 2006, 176, 1686–1694.
[38] A. Phalipon, M. Tanguy, C. Grandjean, C. Guerreiro, F. Belot,D. Cohen, P. J. Sansonetti, L. A. Mulard, J. Immunol. 2009,182, 2241–2247.
[39] F. S. Hassane, A. Phalipon, M. Tanguy, C. Guerreiro, F. Belot,B. Frisch, L. A. Mulard, F. Schuber, Vaccine 2009, 27, 5419–5426.
[40] B. Vulliez-Le Normand, F. A. Saul, A. Phalipon, F. Belot, C.Guerreiro, L. A. Mulard, G. A. Bentley, Proc. Natl. Acad. Sci.USA 2008, 105, 9976–9981.
[41] G. Lemanski, T. Ziegler, Eur. J. Org. Chem. 2006, 2618–2630.[42] a) J. Boutet, L. A. Mulard, Eur. J. Org. Chem. 2008, 5526–5542;
b) J. Boutet, C. Guerreiro, L. A. Mulard, J. Org. Chem. 2009,74, 2651–2670.
[43] Y. Wang, X. Zhang, P. Wang, Org. Biomol. Chem. 2010, 8,4322–4328.
[44] a) B. Benaissa-Trouw, D. J. Lefeber, J. P. Kamerling, J. F. G.Vliegenthart, K. Kraaijeveld, H. Snippe, Infect. Immun. 2001,69, 4698–4701; b) F. Mawas, J. Niggemann, C. Jones, M. J.Corbel, J. P. Kamerling, J. F. G. Vliegenthart, Infect. Immun.2002, 70, 5107–5114.
[45] D. Safari, H. A. T. Dekker, J. A. F. Joosten, D. Michalik, A.Carvalho de Souza, R. Adamo, M. Lahmann, A. Sundgren, S.Oscarson, J. P. Kamerling, H. Snippe, Infect. Immun. 2008, 76,4615–4623.
[46] a) A. R. Parameswar, P. Pornsuriyasak, N. A. Lubanowski,A. V. Demchenko, Tetrahedron 2007, 63, 10083–10091; b) A. R.Parameswar, S. J. Hasty, A. V. Demchenko, Carbohydr. Res.2008, 343, 1707–1717.
[47] A. R. Parameswar, I. H. Park, R. Saksena, P. Kovác, M. H.Nahm, A. V. Demchenko, ChemBioChem 2009, 10, 2893–2899.
[48] E. Bousquet, M. Khitri, L. Lay, F. Nicotra, L. Panza, G.Russo, Carbohydr. Res. 1998, 311, 171–181.
[49] a) T. Sugawara, K. Igarashi, Carbohydr. Res. 1988, 172, 195–207; b) H. Paulsen, B. Helpap, J. P. Lorentzen, Carbohydr. Res.1988, 179, 173–197; c) E. Kaji, F. W. Lichtenthaler, Y. Osa, S.Zen, Bull. Chem. Soc. Jpn. 1995, 68, 2401–2408.
[50] a) G. Soliveri, A. Bertolotti, L. Panza, L. Poletti, C. Jones, L.Lay, J. Chem. Soc. Perkin Trans. 1 2002, 2174–2181; b) M. Nils-son, T. Norberg, J. Chem. Soc. Perkin Trans. 1 1998, 1699–1704.
[51] L. Legnani, S. Ronchi, S. Fallarini, G. Lombardi, F. Campo,L. Panza, L. Lay, L. Poletti, L. Toma, F. Ronchetti, F. Com-postella, Org. Biomol. Chem. 2009, 7, 4428–4436.
[52] V. Pozsgay, J. Kubler-Kielb, B. Coxon, G. Ekborg, Tetrahedron2005, 61, 10470–10481.
[53] V. Pozsgay, J. Kubler-Kielb, Carbohydr. Res. 2007, 342, 621–626.
[54] G. Stübs, B. Rupp, R. R. Schumann, N. W. J. Schröder, J. Rad-emann, Chem. Eur. J. 2010, 16, 3536–3544.
[55] C. Jones, Carbohydrates Europe 1998, 21, 10–16.[56] A. Berkin, B. Coxon, V. Pozsgay, Chem. Eur. J. 2002, 8, 4424–
4433].[57] R. Slättegård, P. Teodorovic, H. Hadgu Kinfe, N. Ravenscroft,
D. W. Gammon, S. Oscarson, Org. Biomol. Chem. 2005, 3,3782–3787.
L. Morelli, L. Poletti, L. LayMICROREVIEW[58] P. Teodorovic, R. Slättegård, S. Oscarson, Org. Biomol. Chem.
2006, 4, 4485–4490.[59] a) M.-I. Torres-Sanchez, V. Draghetti, L. Panza, L. Lay, G.
Russo, Synlett 2005, 1147–1151; b) M.-I. Torres-Sanchez, C.Zaccaria, B. Buzzi, G. Miglio, G. Lombardi, L. Polito, G.Russo, L. Lay, Chem. Eur. J. 2007, 13, 6623–6635.
[60] F. Manea, C. Bindoli, S. Polizzi, L. Lay, P. Scrimin, Langmuir2008, 24, 4120–4124.
[61] F. Manea, C. Bindoli, S. Fallarini, G. Lombardi, L. Polito, L.Lay, R. Bonomi, F. Mancin, P. Scrimin, Adv. Mater. 2008, 20,4348–4352.
[62] a) J. A. Zang, P. Kovác, Carbohydr. Res. 1999, 321, 157–167;b) J. A. Zang, P. Kovác, Bioorg. Med. Chem. Lett. 1999, 9, 487–490; c) R. Saksena, X. Ma, P. Kovác, Carbohydr. Res. 2003,338, 2591–2603; d) R. Saksena, X. Ma, T. K. Wade, P. Kovác,W. F. Wade, Carbohydr. Res. 2005, 340, 2256–2269.
[63] A. Chernyak, S. Kondo, T. K. Wade, M. D. Meeks, P. M. Alz-ari, J. M. Fournier, R. K. Taylor, P. Kovác, W. F. Wade, J. In-fect. Dis. 2002, 185, 950–962.
[64] R. Saksena, J. Zhang, P. Kovác, Tetrahedron: Asymmetry 2005,16, 187–197.
[65] T. K. Wade, R. Saksena, J. Shiloach, P. Kovác, W. F. Wade,FEMS Immunol. Med. Microbiol. 2006, 48, 237–251.
[66] A. Adamo, P. Kovác, Eur. J. Org. Chem. 2007, 988–1000.[67] J. Zhang, P. Kovác, Carbohydr. Res. 1997, 300, 329–339.[68] a) B. Ruttens, P. Kovác, Carbohydr. Res. 2006, 341, 1077–1080;
b) B. Ruttens, R. Saksena, P. Kovác, Eur. J. Org. Chem. 2007,4366–4375.
[69] D. Turek, A. Sundgren, M. Lahmann, S. Oscarson, Org. Bio-mol. Chem. 2006, 4, 1236–1241.
[70] J. C. Sacchettini, E. J. Rubin, J. S. Freundlich, Nat. Rev. Micro-biol. 2008, 6, 41–52.
[71] Y. A. W. Skeiky, J. C. Sadoff, Nat. Rev. Microbiol. 2006, 4, 469–476.
[72] B. Cao, S. Williams, J. Nat. Prod. Rep. 2010, 27, 919–947, andreferences cited therein.
[73] B. H. Hamasur, M. Haile, A. Pawlowski, U. Schroder, A. Wil-liams, G. Hatch, P. Marsh, G. Kallenius, S. B. Svenson, Vaccine2003, 21, 4081–4093.
[74] X. Liu, B. L. Stocker, P. H. Seeberger, J. Am. Chem. Soc. 2006,128, 3638–3648.
[75] a) K. N. Jayaprakash, J. Lu, B. Fraser-Reid, Angew. Chem.2005, 117, 6044; Angew. Chem. Int. Ed. 2005, 44, 5894–5898;b) B. Fraser-Reid, S. R. Chaudhuri, K. N. Jayaprakash, J. Lu,C. V. S. Ramamurty, J. Org. Chem. 2008, 73, 9732–9743.
[76] A. Hölemann, B. L. Stocker, P. H. Seeberger, J. Org. Chem.2006, 71, 8071–8088.
[77] B. Fraser-Reid, J. Lu, K. N. Jayaprakash, J. C. Lopez, Tetrahe-dron: Asymmetry 2006, 17, 2449–2463.
[78] M. Joe, Y. Bai, R. C. Nacario, T. L. Lowary, J. Am. Chem. Soc.2007, 129, 9885–9901.
[79] S. Boonyarattanakalin, X. Liu, M. Michieletti, B. Lepenies,P. H. Seeberger, J. Am. Chem. Soc. 2008, 130, 16792–16799.
[80] J. C. Paulson, O. Blixt, B. E. Collins, Nature Chem. Biol. 2006,2, 238–248.
[81] a) G. De Libero, L. Mori, Nat. Rev. Immunol. 2005, 5, 485–496; b) T. B. H. Geijtenbeek, S. J. van Vliet, E. A. Koppel, M.Sanchez-Hernandez, C. M. J. E. Vandenbroucke-Grauls, B.Appelmelk, Y. van Kooyk, J. Exp. Med. 2007, 197, 7–17.
[82] J. Guiard, A. Collmann, M. Gilleron, L. Mori, G. De Libero,J. Prandi, G. Puzo, Angew. Chem. 2008, 120, 1; Angew. Chem.Int. Ed. 2008, 47, 1–6.
[83] There is a licensed vaccine for the prevention of anthrax, butit is not available for general use.
[84] D. B. Werz, P. H. Seeberger, Angew. Chem. 2005, 117, 6474;Angew. Chem. Int. Ed. 2005, 44, 6315–6318.
[85] M. Tamborrini, D. B. Werz, J. Frey, G. Pluschke, P. H. Seeb-erger, Angew. Chem. 2006, 118, 6731; Angew. Chem. Int. Ed.2006, 45, 6581–6582.
[86] D. B. Werz, A. Adibekian, P. H. Seeberger, Eur. J. Org. Chem.2007, 1976–1982.
[87] a) R. Adamo, R. Saksena, P. Kovác, Carbohydr. Res. 2005, 340,2579–2582; b) R. Adamo, R. Saksena, P. Kovác, Helv. Chim.Acta 2006, 89, 1074–1089; c) R. Saksena, R. Adamo, P. Kovác,Bioorg. Med. Chem. 2007, 15, 4283–4310.
[88] S. Hou, P. Kovác, Synthesis 2009, 545–550.[89] A. S. Metha, E. Saile, W. Zhong, T. Buskas, R. Carlson, E.
Kannenberg, Y. Reed, C. Quinn, G.-J. Boons, Chem. Eur. J.2006, 12, 9136–9149.
[90] a) I. Braccini, C. Derouet, J. Esnault, C. H. du Penhoat, J. M.Mallet, V. Michon, P. Sinaÿ, Carbohydr. Res. 1993, 246, 23–41;b) Y. D. Vankar, P. S. Vankar, M. Behrendt, R. R. Schmidt,Tetrahedron 1991, 47, 9985–9992.
[91] D. Crich, O. Vinogradova, J. Org. Chem. 2007, 72, 6513–6520.[92] S. G. Y. Dhénin, V. Moreau, N. Morel, M.-C. Nevers, H. Vol-
land, C. Créminon, F. Djedaïni-Pilard, Carbohydr. Res. 2008,343, 2101–2110.
[93] a) H. Guo, G. A. O’Doherty, Angew. Chem. 2007, 119, 5298;Angew. Chem. Int. Ed. 2007, 46, 5206–5208; b) H. Guo, G. A.O’Doherty, J. Org. Chem. 2008, 73, 5211–5220.
[94] O. Achmatowicz, R. Bielski, Carbohydr. Res. 1977, 55, 165–176.
[95] R. S. Babu, G. A. O’Doherty, J. Am. Chem. Soc. 2003, 125,12406–12407.
[96] M. Vasan, J. Rauvolfova, M. A. Wolfert, C. Leoff, E. L. Kan-nenberg, C. P. Quinn, R. W. Carlson, G.-J. Boons, ChemBio-Chem 2008, 9, 1716–1720.
[97] M. A. Oberli, P. Bindschädler, D. B. Werz, P. H. Seeberger, Org.Lett. 2008, 10, 905–908.
[98] A. H. Reid, J. K. Taubenberger, T. G. Fanning, Microbes Infect.2001, 3, 81–87.
[99] R. B. Belshe, Virus Res. 2004, 103, 177–185.[100] D. J. Vigerust, V. L. Shepherd, Trends Microbiol. 2007, 15,
211–218.[101] M. S. Gottlieb, R. Schroff, H. M. Schenker, J. D. Weisman,
P. T. Fan, R. A. Wolf, A. Saxon, N. Engl. J. Med. 1981, 305,1425–1431.
[102] a) F. Barré-Sinoussi, J. C. Chermann, F. Rey, M. T. Nugeyre,S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, L. Montagnier, Science1983, 220, 868–871; b) M. Popovic, M. G. Sarngadharan, Sci-ence 1984, 224, 497–500; c) J. A. Levy, A. D. Hoffmann, S.Kramer, J. A. Landis, J. M. Shimabukuro, L. S. Oshiro, Sci-ence 1984, 225, 840–842.
[103] F. Clavel, K. Mansinho, S. Chamaret, D. Guetard, V. Favier,J. Nina, M.-O. Santos-Ferreira, J.-L. Champalimaud, L.Montagnier, N. Engl. J. Med. 1987, 316, 1180–1185.
[104] R. Marlink, P. Kanki, I. Thior, K. Travers, G. Eisen, T. Siby,I. Traore, C.-C. Hsieh, M. C. Dia, E.-H. Gueye, J. Hellinger,A. Gueye-Ndiaye, J.-L. Sankale, I. Ndoye, S. Mboup, M. Es-sex, Science 1994, 265, 1587–1590.
[105] a) P. Poumbourios, K. A. Wilson, R. J. Center, W. El Ahmar,B. E. Kemp, J. Virol. 1997, 71, 2041–2049; b) D. C. Chan, D.Fass, J. M. Berger, P. S. Kim, Cell 1997, 89, 263–273; c) W.Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, D. C.Wiley, Nature 1997, 387, 426–430.
[106] D. M. Eckert, P. S. Kim, Annu. Rev. Biochem. 2001, 70, 777–810.
[107] a) R. Wyatt, P. D. Kwong, E. Desjardins, R. W. Sweet, J. Rob-inson, W. A. Hendrickson, J. G. Sodroski, Nature 1998, 393,705–711; b) P. Poignard, E. O. Saphire, P. W. Parren, D. R.Burton, Annu. Rev. Immunol. 2001, 19, 253–274.
[108] B. Korber, B. Gaschen, K. Yusim, R. Thakallapally, C. Kes-mir, V. Detours, Br. Med. Bull. 2001, 58, 19–42.
[109] a) H. Li, L.-X. Wang, Org. Biomol. Chem. 2004, 2, 483–488;b) R. Pantophlet, D. R. Burton, Annu. Rev. Immunol. 2006,24, 739–769; c) A. J. Conley, J. A. Kessler, L. J. Boots, J. S.Tung, B. A. Arnold, P. M. Keller, A. R. Shaw, E. A. Emini,Proc. Natl. Acad. Sci. USA 1994, 91, 3348–3352.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
[110] a) C. E. Parker, L. J. Deterding, C. Hager-Braun, J. M. Binley,N. Schulke, H. Katinger, J. P. Moore, K. B. Tomer, J. Virol.2001, 75, 10906–10911; b) M. B. Zwick, A. F. Labrijn, M.Wang, C. Spenlehauer, E. O. Saphire, J. M. Binley, J. P.Moore, G. Stiegler, H. Katinger, D. R. Burton, P. W. Parren,J. Virol. 2001, 75, 10892–10905; c) G. Stiegler, R. Kunert, M.Purtscher, S. Wolbank, R. Voglauer, F. Steindl, H. Katinger,AIDS Res. Hum. Retroviruses 2001, 17, 1757–1765.
[111] a) D. R. Burton, J. Pyati, R. Koduri, S. J. Sharp, G. B. Thorn-ton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop,P. L. Nara, M. Lamacchia, E. Garratty, E. R. Stiehm, Y. J.Bryson, J. P. Moore, D. D. Ho, C. F. Barbas, Science 1994,266, 1024–1027; b) E. O. Saphire, P. W. Parren, R. Pantophlet,M. B. Zwick, G. M. Morris, P. M. Rudd, R. A. Dwek, R. L.Stanfield, D. R. Burton, I. A. Wilson, Science 2001, 293,1155–1159.
[112] D. A. Calarese, C. N. Scanlan, M. B. Zwick, S. Deechongkit,Y. Mimura, R. Kunert, P. Zhu, M. R. Wormald, R. L. Stan-field, K. H. Roux, J. W. Kelly, P. M. Rudd, R. A. Dwek, H.Katinger, D. R. Burton, I. A. Wilson, Science 2003, 300,2065–2070.
[113] D. A. Calarese, H.-K. Lee, C.-Y. Huang, M. D. Best, R. D.Astronomo, R. L. Stanfield, H. Katinger, D. R. Burton, C.-H. Wong, I. A. Wilson, Proc. Natl. Acad. Sci. USA 2005, 102,13372–13377.
[114] C. N. Scanlan, J. Offer, N. Zitzmann, R. A. Dwek, Nature2007, 446, 1038–1045.
[115] a) L.-X. Wang, J. Ni, S. Singh, H. Li, Chem. Biol. 2004, 2,483–488; b) J. Ni, H. Song, Y. Wang, N. M. Stamatos, L.-X.Wang, Bioconjugate Chem. 2006, 17, 493–500; c) J. Wang, H.Li, G. Zou, L.-X. Wang, Org. Biomol. Chem. 2007, 5, 1529–1540; d) V. Y. Dudkin, M. Orlova, X. Geng, M. Mandal,W. C. Olson, S. J. Danishefsky, J. Am. Chem. Soc. 2004, 126,9560–9562; e) M. Mandal, V. Y. Dudkin, X. Geng, S. Danish-efsky, Angew. Chem. 2004, 116, 2611; Angew. Chem. Int. Ed.2004, 43, 2557–2561; f) X. Geng, V. Y. Dudkin, M. Mandal,S. Danishefsky, Angew. Chem. Int. Ed. 2004, 43, 2552–2565.
[116] H. Lis, N. Sharon, J. Biol. Chem. 1978, 253, 3468–3476.[117] a) H. Li, L. X. Wang, Org. Biomol. Chem. 2003, 1, 3507–3513;
b) J. Ni, S. Singh, L.-X. Wang, Bioorg. Med. Chem. 2003, 11,159–166; c) J. Ni, S. Singh, L.-X. Wang, Bioconjugate Chem.2003, 14, 232–238.
[118] a) G. Tuchscherer, C. Servis, G. Corradin, U. Blum, J. Rivier,M. Mutter, Protein Sci. 1992, 1, 1377–1386; b) G. Tuchsch-erer, D. Grell, M. Mathieu, M. Mutter, J. Pept. Res. 1999, 54,185–194; c) S. Peluso, T. Ruckle, C. Lehmann, M. Mutter, C.Peggion, M. Crisma, ChemBioChem 2001, 2, 432–437; d) O.Renaudet, P. Dumy, Org. Lett. 2003, 5, 243–246.
[119] H.-K. Lee, C. N. Scanlan, C.-Y. Huang, A. Y. Chang, D. A.Calarese, R. A. Dwek, P. M. Rudd, D. R. Burton, I. A. Wil-son, C.-H. Wong, Angew. Chem. 2004, 116, 1018; Angew.Chem. Int. Ed. 2004, 43, 1000–1003.
[120] J. D. Warren, X. Geng, S. J. Danishefsky, Top. Curr. Chem.2007, 267, 109–141.
[121] I. J. Krauss, J. G. Joyce, A. C. Finnefrock, H. C. Song, V. Y.Dudkin, X. Geng, J. D. Warren, M. Chastain, J. W. Shiver,S. J. Danishefsky, J. Am. Chem. Soc. 2007, 129, 11042–11044.
[122] a) P. Dumy, I. M. Eggleston, S. Cervigni, U. Sila, X. Sun, M.Mutter, Tetrahedron Lett. 1995, 36, 1225–1258; b) P. Dumy,O. Renaudet, Org. Lett. 2003, 5, 243–246; c) Y. Singh, G. T.Dolphin, J. Razkin, P. Dumy, ChemBioChem 2006, 7, 1298–1314.
[123] a) L. Jiang, K. Moehle, B. Dhanapal, D. Obrecht, J. A. Rob-inson, Helv. Chim. Acta 2000, 83, 3097–3112; b) M. Favre, K.Moehle, L. Jiang, B. Pfeiffer, J. A. Robinson, J. Am. Chem.Soc. 1999, 121, 2679–2685; c) J. A. Robinson, Synlett 2000,4, 429–441.
[124] J. W. Bean, K. D. Kopple, C. E. Peishoff, J. Am. Chem. Soc.1992, 114, 5328–5334.
[125] T. Cohen-Anisfeld, P. T. Lansbury, J. Am. Chem. Soc. 1993,115, 10531–10537.
[126] a) G. B. McGaughey, M. Citron, R. C. Danzeisen, R. M. Fre-idinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M.Miller, J. Shiver, M. J. Bogusky, Biochemistry 2003, 42, 3214–3223; b) J. Joyce, J. Cook, D. Chabot, R. Hepler, W. Shoop,Q. Xu, T. Stambaugh, M. Aste-Amezaga, S. Wang, L. Indra-wati, M. Bruner, A. Friedlander, P. Keller, M. Caulfield, J.Biol. Chem. 2006, 281, 4831–4843.
[127] Z. Y. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, C.-H.Wong, J. Am. Chem. Soc. 1999, 121, 734–753.
[128] E. W. Adams, D. M. Ratner, H. R. Bokesch, J. B. McMahon,B. R. O’Keefe, P. H. Seeberger, Chem. Biol. 2004, 11, 875–881.
[129] S.-K. Wang, P.-H. Liang, R. D. Astronomo, T.-L. Hsu, S.-L.Hsieh, D. R. Burton, C.-H. Wong, Proc. Natl. Acad. Sci. USA2008, 105, 3690–3695.
[130] T. B. H. Geijtenbeek, D. S. Kwon, R. Torensma, S. J.Van Vliet, G. C. F. van Duijnhoven, J. Middel, I. L. Cor-nelissen, H. S. Nottet, V. N. Kewal Ramani, D. R. Littman,C. G. Figdor, Y. van Kooyk, Cell 2000, 100, 587–597.
[131] K. Gorska, K.-T. Huang, O. Chaloin, N. Winssinger, Angew.Chem. 2009, 121, 7831; Angew. Chem. Int. Ed. 2009, 48, 7695–7700.
[132] Y. M. Chabre, R. Roy, Curr. Top. Med. Chem. 2008, 8, 1237–1285.
[133] A. Kabanova, R. Adamo, D. Proietti, F. Berti, M. Tontini,R. Rappuoli, P. Costantino, Glycoconjugate J. 2010, 27, 501–513.
[134] R. D. Astronomo, E. Kaltgrad, A. K. Udit, S.-K. Wang, K. J.Doores, C.-Y. Huang, R. Pantophlet, J. C. Paulson, C.-H.Wong, M. G. Finn, D. R. Burton, Chem. Biol. 2010, 17, 357–370.
[135] K. J. Doores, Z. Fulton, V. Hong, M. K. Patela, C. N. Scan-lang, M. R. Wormaldg, M. G. Finn, D. R. Burton, I. A. Wil-son, B. G. Davis, Proc. Natl. Acad. Sci. USA 2010, 107,17107–17112.
[136] V. Hong, S. I. Presolski, C. Ma, M. G. Finn, Angew. Chem.2009, 121, 10063; Angew. Chem. Int. Ed. 2009, 48, 9879–9883.
[137] a) R. G. Webster, in: Options for the Control of InfluenzaVirus II (Ed.: C. Hannoun), Excerpta Medica, Amsterdam,1993, pp. 177–185; b) R. Wagner, M. Matrosovich, H.-D.Klenk, Rev. Med. Virol. 2002, 12, 159–166.
[138] a) K. Subbarao, A. Klimov, J. Katz, H. Regnery, W. Lim, H.Hall, M. Perdue, D. Swayne, C. Bender, J. Huang, M. Hem-phill, T. Rowe, M. Shaw, X. Xu, K. Fukuda, N. Cox, Science1998, 279, 393–396; b) M. Peiris, K. Y. Yuen, C. W. Leung,K. H. Chan, P. L. S. Ip, R. W. M. Lai, K. W. Orr, K. F.Shortridge, Lancet 1999, 354, 916–917.
[139] R. J. Webby, R. Webster, Science 2003, 302, 1519–1522.[140] a) J. J. Skehel, D. C. Wiley, Annu. Rev. Biochem. 2000, 69,
531–569; b) J. N. Couceiro, J. C. Paulson, L. G. Baum, VirusRes. 1993, 29, 155–165; Y. Suzuki, T. Ito, T. Suzuki, R. E.Holland Jr, T. M. Chambers, M. Kiso, H. Ihida, Y. Kawaoka,J. Virol. 2000, 74, 11825–11831.
[142] a) I. A. Wilson, J. J. Skehel, D. C. Wiley, Nature 1981, 289,366–373; b) W. Weis, J. H. Brown, S. Cusack, J. C. Paulson,J. J. Skehel, D. C. Wiley, Nature 1988, 333, 426–431; c) N. K.Sauter, G. D. Glick, R. L. Crowther, S.-J. Park, M. B. Eisen,J. J. Skehel, J. R. Knowles, D. C. Wiley, Proc. Natl. Acad. Sci.USA 1992, 89, 324–328; d) S. J. Watowich, J. J. Skehel, D. C.Wiley, Structure 1994, 2, 719–731.
[143] D. C. Wiley, J. J. Skehel, Annu. Rev. Biochem. 1987, 56, 365–394.
[144] R. Wagner, M. Matrosovich, H. Klenk, Rev. Med. Virol. 2002,12, 159–166.
[145] H.-D. Klenk, R. Wagner, D. Heuer, T. Wolff, Virus Res. 2002,82, 73–75.
L. Morelli, L. Poletti, L. LayMICROREVIEW[146] a) R. Daniels, B. Kurowski, A. E. J. N. Hebert, Mol. Cell
2003, 11, 79–90; b) R. Wagner, T. Wolff, A. Herwig, J. Virol.2000, 74, 6316–6323; c) S. J. Baignet, J. W. McCauley, VirusRes. 2001, 79, 177–185; d) E. Tsuchiya, K. Sugawara, S.Hongo, Y. Matsuzaki, Y. Muraki, Z.-N. Li, K. Nakamura, J.Gen. Virol. 2002, 83, 3067–3074; e) E. Tsuchiya, K. Sugawara,S. Hongo, Y. Matsuzaki, Y. Muraki, Z.-N. Li, K. Nakamura,J. Gen. Virol. 2002, 83, 1137–1146; f) Y. Abe, T. Takashita,K. Sugawara, Y. Matsuzaki, Y. M. Hongo, J. Virol. 2004, 78,9605–9611; g) N. V. Kaverin, I. A. Rudneva, N. A. Ilyushima,N. L. Varich, A. S. Lipatov, Y. A. Smirnov, E. A. Govorkova,A. K. Gitelman, D. K. Lvov, R. G. Webster, J. Gen. Virol.2002, 83, 2497–2505; h) H. Segawa, A. Inakawa, T. Yamash-ita, H. Taira, Biosci. Biotechnol. Biochem. 2003, 67, 592–598;i) J. J. Skehel, D. J. Stevens, R. S. Daniels, A. R. Douglas, M.Knossow, I. A. Wilson, D. C. Wiley, Proc. Natl. Acad. Sci.USA 1984, 81, 1779–1783.
[147] a) P. J. Gallagher, J. M. Henneberry, J. F. Sambrook, M. J. Ge-thing, J. Virol. 1992, 66, 7136–7145; b) P. C. Roberts, W.Garten, H.-D. Klenk, J. Virol. 1993, 67, 3048–3060; c) E. No-busawa, T. Aoyama, H. Kato, Y. Suzuki, Y. Tateno, K. Naka-jima, Virology 1991, 182, 475–485.
[148] M. Igarashi, K. Ito, H. Kida, A. Takada, Virology 2008, 376,323–329.
[149] D. Vigerust, K. Ulett, K. Boyd, J. Madsen, S. Hawgood, J.McCullers, J. Virol. 2007, 81, 8593–8600.
[150] P. Gallagher, J. Henneberry, I. Wilson, J. Sambrook, M. Ghet-ing, J. Cell. Biol. 1988, 107, 2059–2073.
[151] a) K. L. Deshpande, V. A. Fried, M. Ando, R. G. Webster,Proc. Natl. Acad. Sci. USA 1987, 84, 36–40; b) M. Ohuchi,R. Ohuchi, A. Feldmann, H. Klenk, J. Virol. 1997, 71, 8377–8384; c) R. Wagner, D. Heuer, T. Wolff, A. Herwig, H.-D.Klenk, J. Gen. Virol. 2002, 83, 601–609; d) R. Ohuchi, M.Ohuchi, W. Garten, H. Klenk, J. Virol. 1997, 71, 3719–3725.
[152] R. G. Webster, Vaccine 2000, 18, 1686–1689.[153] U. M. Abdel-Motal, H. M. Guay, K. Wigglesworth, R. M.
Welsh, U. Galili, J. Virol. 2007, 81, 9131–9141.[154] U. Galili, E. Rachmilewitz, A. Peleg, I. Flechner, J. Exp. Med.
1984, 160, 1519–1531.[155] U. Galili, Springer Semin. Immunopathol. 1993, 15, 155–171.[156] T. R. Henion, W. Gerhard, F. Anaraki, U. Galili, Vaccine
1997, 15, 1174–1182.[157] U. Abdel-Motal, S. Wang, S. Lu, K. Wigglesworth, U. Galili,
J. Virol. 2006, 80, 6943–6951.[158] D. LaTemple, J. Abrams, S. Zhang, U. Galili, Cancer Res.
S.-W. Chen, C.-M. Chen, K.-H. Khoo, T.-J. Cheng, Y.-S. E.Cheng, J.-T. Jan, C.-Y. Wu, C. Ma, C.-H. Wong, Proc. Natl.Acad. Sci. USA 2009, 106, 18137–18142.
[160] a) E. Hoffmann, A. S. Lipatov, R. J. Webby, E. A. Govorkova,R. G. Webster, Proc. Natl. Acad. Sci. USA 2005, 102, 12915–12920; b) J. W. Huleatt, V. Nakaar, P. Desai, Y. Huang, D.Hewitt, A. Jacobs, J. Tang, W. McDonald, L. Song, R. K.Evans, S. Umlauf, L. Tussey, T. J. Powell, Vaccine 2008, 26,201–214; c) C. N. Scanlan, G. E. Ritchie, K. Baruah, M. Cris-pin, D. J. Harvey, B. B. Singer, L. Lucka, M. R. Wormald, P.Wentworth Jr, N. Zitzmann, P. M. Rudd, D. R. Burton, R. A.Dwek, J. Mol. Biol. 2007, 372, 16–22; d) Z. Y. Yang, Z.-Y.Yang, C.-J. Wei, W.-P. Kong, L. Wu, L. Xu, D. F. Smith, G. J.Nabel, Science 2007, 317, 825–828.
[161] a) D. C. Ekiert, G. Bhabha, M.-A. Elsliger, R. H. E. Friesen,M. Jongeneelen, M. Throsby, J. Goudsmit, I. A. Wilson, Sci-ence 2009, 324, 246–251; b) A. K. Kashyap, J. Steel, A. F.Oner, M. A. Dillon, R. E. Swale, K. M. Wall, K. J. Perry, A.Faynboym, M. Ilhan, M. Horowitz, L. Horowitz, P. Palese,R. R. Bhatt, R. A. Lerner, Proc. Natl. Acad. Sci. USA 2008,105, 5986–5991; c) J. F. Scheid, H. Mouquet, N. Feldhahn,M. S. Seaman, K. Velinzon, J. Pietzsch, R. G. Ott, R. M. An-thony, H. Zebroski, A. Hurley, A. Phogat, B. Chakrabarti, Y.
Li, M. Connors, F. Pereyra, B. D. Walker, H. Wardemann, D.Ho, R. T. Wyatt, J. R. Mascola, J. V. Ravetch, M. C. Nus-senzweig, Nature 2009, 458, 636–640; d) J. Stevens, O. Blixt,T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, I. A. Wil-son, Science 2006, 312, 404–410; e) I. Sui, W. C. Hwang, S.Perez, G. Wei, D. Aird, L. Chen, E. Santelli, B. Stec, G.Cadwell, M. Ali, H. Wan, A. Murakami, A. Yammanuru, T.Han, N. J. Cox, L. A. Bankston, R. O. Donis, R. C. Lidding-ton, W. A. Marasco, Nat. Struct. Mol. Biol. 2009, 16, 265–273; f) M. Throsby, E. van den Brink, M. Jongeneelen,L. L. M. Poon, P. Alard, L. Cornelissen, A. Bakker, F. Cox,E. van Deventer, Y. Guan, J. Cinatl, J. ter Meulen, I. Lasters,R. Carsetti, M. Peiris, J. de Kruif, J. Goudsmit, PLoS ONE2008, 3, e3942.
[162] J. B. Dixon, Comp. Immunol. Microbiol. Infect. Dis. 1997, 20,87–94.
[163] A. Kwame Nyame, Z. S. Kawar, R. D. Cummings, Arch. Bio-chem. Biophys. 2004, 426, 182–200.
[164] WHO World Malaria Report 2009 in: http://www.who.int/malaria/world_malaria_report_2009/en/index.html (accessedon September, 27th 2010).
[165] a) S. M. Todryk, A. V. S. Hill, Nature Rev. Microbiol. 2007,5, 487–489; b) J. Langhorne, F. M. Ndungu, A.-M. Sponaas,K. Marsh, Nature Immunol. 2008, 9, 725–732.
[166] D. C. Gowda, E. A. Davidson, Parasitol. Today 1999, 15,147–152.
[167] a) L. Schofield, M. J. McConville, D. Hansen, A. S.Campbell, B. Fraser-Reid, M. J. Grusby, S. D. Tachado, Sci-ence 1999, 283, 225–229; b) I. A. Clark, L. Schofield, Parasi-tol. Today 2000, 16, 451–454.
[168] a) L. Schoefield, M. C. Hewitt, K. Evans, A. Siomos, P. H.Seeberger, Nature 2002, 418, 785–789; b) M. C. Hewit, D. A.Snyder, P. H. Seeberger, J. Am. Chem. Soc. 2002, 124, 13434–13436.
[169] P. H. Seeberger, R. L. Soucy, Y.-U. Kwon, D. A. Snyder, T.Kanemitsu, Chem. Commun. 2004, 1706–1707.
[170] a) R. L. Soucy, D. A. Snyder, P. H. Seeberger, Chem. Eur. J.2005, 11, 2493–2504; b) X. Liu, Y.-U. Kwon, P. H. Seeberger,J. Am. Chem. Soc. 2005, 127, 5004–5005.
[171] F. Kamena, M. Tamborrini, X. Liu, Y.-U. Kwon, F. Thomp-son, G. Pluschke, P. H. Seeberger, Nature Chem. Biol. 2008,4, 238–240.
[172] M. Tamborrini, X. Liu, J. P. Mugasa, Y.-U. Kwon, F. Ka-mena, P. H. Seeberger, G. Pluschke, Bioorg. Med. Chem. 2010,18, 3747–3752.
[173] a) M. J. McConville, M. A. J. Ferguson, Biochem. J. 1993,294, 305–324; b) A. Guha-Niyogi, D. R. Sullivan, S. J. Turco,Glycobiology 2001, 11, 45R–59R.
[174] a) S. J. Turco, G. F. Späth, S. M. Beverley, Trends Parasitol.2001, 17, 223–226; b) G. F. Späth, L.-F. Lye, H. Segawa, D.Sacks, S. J. Turco, S. M. Beverley, Science 2003, 301, 1241–1243; c) A. Düffels, S. V. Ley, J. Chem. Soc. Perkin Trans. 11999, 375–378; d) K. Ruda, J. Lindberg, P. J. Garegg, S. Os-carson, P. Konradsson, Tetrahedron 2000, 56, 3969–975; e) K.Ruda, J. Lindberg, P. J. Garegg, S. Oscarson, P. Konradsson,J. Am. Chem. Soc. 2000, 122, 11067–11072; f) L. Gandolfi-Donadio, C. Gallo-Rodriguez, R. M. de Lederkremer, J. Org.Chem. 2002, 67, 4430–4435; g) D. Majumdar, G. A. Elsayed,T. Buskas, G.-J. Boons, J. Org. Chem. 2005, 70, 1691–1697.
[175] a) S. M. Beverley, S. Turco, J. Trends Microbiol. 1998, 6, 35–40; b) S. J. Turco, A. Descoteaux, Ann. Rev. Microbiol. 1992,46, 65–94; c) M. Kelleher, A. Bacic, E. Handman, Proc. Natl.Acad. Sci. USA 1992, 89, 6–10; d) P. Talamas-Rohana, S. D.Wright, M. R. Lennartz, D. G. Russell, J. Immunol. 1990, 144,4817–4824; e) D. M. Mosser, T. A. Springer, M. S. Diamond,J. Cell. Biol. 1992, 116, 511–520; f) S. W. Homans, A.Mehlert, S. Turco, J. Biochem. 1992, 31, 654–661.
[176] see also: A. J. Ross, O. V. Sizova, A. V. Nikolaev, Carbohydr.Res. 2006, 341, 1954–1964.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
[177] a) A. V. Nikolaev, T. J. Rutherford, M. A. J. Ferguson, J. S. J.Brimacombe, J. Chem. Soc. Perkin Trans. 1 1995, 1977–1987;b) A. V. Nikolaev, T. J. Rutherford, M. A. J. Ferguson, J. S. J.Brimacombe, J. Chem. Soc. Perkin Trans. 1 1996, 1559–1566;c) A. P. Higson, Y. E. Tsvetov, M. A. J. Ferguson, A. V. Niko-laev, J. Chem. Soc. Perkin Trans. 1 1998, 2587–2595; d) A. P.Higson, A. J. Ross, Y. E. Tsvetkov, F. H. Routier, O. V. Sizova,M. A. J. Ferguson, A. V. Nikolaev, Chem. Eur. J. 2005, 11,2019–2030.
[178] F. H. Routier, A. V. Nikolaev, M. A. J. Ferguson, Glycocon-jugate J. 1999, 16, 773–780.
[179] M. E. Rogers, O. V. Sizova, M. A. J. Ferguson, A. V. Nikolaev,P. A. Bates, J. Infect. Dis. 2006, 194, 512–518.
[180] V. S. Borodkin, F. C. Milne, M. A. J. Ferguson, A. V. Niko-laev, Tetrahedron Lett. 2002, 43, 7821–7825.
[181] a) R. Engel, Chem. Rev. 1977, 77, 349–367; b) K.-H. Jung,R. R. Schmidt, in: Carbohydrate-Based Drug Discovery, vol. 2(Ed.: C.-H. Wong), Wiley-VCH, Weinheim, Germany, 2003,pp. 609–659.
[182] M. Upreti, R. A. Vishwakarma, Tetrahedron Lett. 1999, 40,2619–2622.
[183] D. Ruhela, R. A. Vishwakarma, Chem. Commun. 2001, 2024–2025.
[184] D. Ruhela, R. A. Vishwakarma, J. Org. Chem. 2003, 68,4446–4456.
[185] M. C. Hewitt, P. H. Seeberger, J. Org. Chem. 2001, 66, 4233–4243.
[186] X. Liu, S. Siegrist, M. Amacker, R. Zurbriggen, G. Pluschke,P. H. Seeberger, ACS Chem. Biol. 2006, 1, 161–164.
[187] D. H. Molyneux, in: Trypanosomiasis and Leishmaniasis(Eds.: G. Hide et al.), CAB International, Wallingford, 1997,pp. 39–50.
[188] I. C. Almeida, R. T. Gazzinelli, J. Leukocyte Biol. 2001, 70,467.
[189] See, for instance: a) V. M. Mendoza, G. A. Kashiwagi, R. M.de Lederkremer, C. Gallo-Rodriguez, Carbohydr. Res. 2010,345, 385–396; b) R. M. van Well, B. Y. M. Collet, R. A. Field,Synlett 2008, 14, 2175–2177 and references cited therein.
[190] M. Hederos, P. Konradsson, J. Am. Chem. Soc. 2006, 128,3414–3419.
[191] M. Hederos, P. Konradsson, J. Org. Chem. 2005, 70, 7196–7207.
[192] D. V. Yashunsky, V. S. Borodkin, M. A. J. Ferguson, A. V. Ni-kolaev, Angew. Chem. 2006, 118, 482; Angew. Chem. Int. Ed.2006, 45, 468–474.
[193] B. Striepen, C. F. Zinecker, J. B. L. Damm, P. A. T. Melgers,G. J. Gerwing, M. Koolen, J. F. G. Vliegenthart, J.-F. Dubre-metz, R. T. Schwarz, J. Mol. Biol. 1997, 266, 797–813.
[194] B. Striepen, J.-F. Dubremetz, R. T. Schwarz, Biochemistry1999, 38, 1479–1487.
[195] K. Pekari, D. Tailler, R. Weingart, R. R. Schmidt, J. Org.Chem. 2001, 66, 7432–7442.
[196] Y.-U. Kwon, X. Liu, P. H. Seeberger, Chem. Commun. 2005,2280–2282.
[197] a) F. Debierre-Grockiego, N. Azzouz, J. Schmidt, J.-F. Dubre-metz, H. Geyer, R. Geyer, R. Weingart, R. R. Schmidt, R. T.Schwartz, J. Biol. Chem. 2003, 278, 32987–32993; b) F. Debi-erre-Grockiego, M. A. Campos, N. Azzouz, J. Schmidt, U.Bieker, M. Garcia Resende, D. Santos Mansur, R. Weingart,R. R. Schmidt, D. Golembock, R. T. Gazzinelli, R. T.Schwartz, J. Immunol. 2007, 179, 1129–1137.
[198] A.-M. M. van Roon, B. Aguilera, F. Cuenca, A. van Remoor-tere, G. A. van der Marel, A. M. Deelder, H. S. Overkleef,C. H. Hokke, Bioorg. Med. Chem. 2005, 13, 3553–3564.
[199] Some examples can be found in: a) A. Carvalho de Souza, J.Kuil, C. E. P. Maljaars, K. M. Halkes, J. F. G. Vliegenthart,J. P. Kamerling, Org. Biomol. Chem. 2004, 2, 2972–2987; b)K. Ágoston, J. Kerékgyártó, J. Hajkó, G. Batta, D. J. Lefeber,J. P. Kamerling, J. F. G. Vliegenthart, Chem. Eur. J. 2002, 8,
151–161; c) J. Nakano, A. Ishiwata, H. Ohta, Y. Ito, Car-bohydr. Res. 2007, 342, 675–695.
[200] T. Yamamura, N. Hada, A. Kaburaki, K. Yamano, T. Takeda,Carbohydr. Res. 2004, 339, 2749–2759.
[201] A. Koizumi, N. Hada, A. Kaburaki, K. Yamano, F.Schweizer, T. Takeda, Carbohydr. Res. 2009, 344, 856–868.
[202] a) P. Zhang, J. Appleton, C.-C. Ling, D. R. Bundle, Can. J.Chem. 2002, 80, 1141–1161; b) M. Nitz, D. R. Bundle, J. Org.Chem. 2000, 65, 3064–3073.
[203] a) H. Goodridge, A. J. Wolf, D. M. Underhill, Immunol. Rev.2009, 230, 38–50; b) E. L. Adams, J. Pharmacol. Exp. Ther.2008, 325, 115–123.
[204] J. E. Cutler, G. S. Deepe Jr, B. S. Klein, Nature Rev. Micro-biol. 2005, 5, 13–28, and references cited therein.
[205] a) F. Fleury, J. Clin. Microbiol. Rev. 1981, 9, 335–348; b) J. D.Sobel, Clin. Infect. Dis. 1992, 14 (Suppl. 1), S148–S153.
[206] A. A. Karelin, Y. E. Tsvetkov, L. Paulovicová, S. Bystrický,E. Paulovicová, N. E. Nifantiev, Carbohydr. Res. 2009, 344,29–35.
[207] a) Y. Han, J. E. Cutler, Immunobiology 1997, 63, 2714–2719;b) Y. Han, M. H. Riessleman, J. E. Cutler, Infect. Immun.2000, 68, 1649–1654; c) Y. Han, M. A. Ulrich, J. E. Cutler, J.Infect. Dis. 1999, 179, 1477–1484.
[208] D. Crich, S. Sun, J. Org. Chem. 1996, 61, 4506–4507, andreferences cited therein.
[209] a) M. Nitz, B. W. Purse, D. R. Bundle, Org. Lett. 2000, 2,2939–2942; b) M. Nitz, D. R. Bundle, J. Org. Chem. 2001, 66,8411–8423.
[210] M. Nitz, C.-C. Ling, A. Ottler, J. E. Cutler, D. R. Bundle, J.Biol. Chem. 2002, 277, 3440–3446.
[211] X. Wu, D. R. Bundle, J. Org. Chem. 2005, 70, 7381–7388.[212] X. Wu, T. Lipinski, F. R. Carrel, J. J. Baileya, D. R. Bundle,
Org. Biomol. Chem. 2007, 5, 3477–3485.[213] S. Dziadek, S. Jacques, D. R. Bundle, Chem. Eur. J. 2008, 14,
5908–5917.[214] H. Xin, S. Dziadek, D. R. Bundle, J. E. Cutler, Proc. Natl.
Acad. Sci. USA 2008, 105, 13526–13531.[215] D. R. Bundle, J. R. Rich, S. Jacques, H. N. Yu, M. Nitz, C.-
[216] X. Wu, T. Lipinski, E. Paszkiewicz, D. R. Bundle, Chem. Eur.J. 2008, 14, 6474–6482.
[217] C. M. Nycholat, D. R. Bundle, Carbohydr. Res. 2009, 344,1397–1411.
[218] J. R. Perfect, A. Casadevall, Infect Dis. Clin. North Am. 2002,16, 837–874.
[219] Y. C. Chang, K. J. Kwon-Chung, Mol. Cell. Biol. 1994, 14,4912–4919.
[220] D. E. Wilson, J. E. Bennett, J. W. Bailey, Proc. Soc. Exp. Biol.Med. 1968, 127, 820.
[221] T. R. Kozel, C. A. Hermerath, Infect. Immun. 1984, 43, 879–886.
[222] L. Pirofski, Trends Microbiol. 2001, 9, 445–451.[223] a) P. J. Garegg, L. Olsson, S. Oscarson, J. Carbohydr. Chem.
1993, 12, 195–209; b) P. J. Garegg, L. Olsson, S. Oscarson,Bioorg. Med. Chem. 1996, 4, 1867–1871.
[224] a) J. Zhang, F. Kong, Carbohydr. Res. 2003, 338, 1719–1725;b) J. Zhang, F. Kong, Tetrahedron Lett. 2003, 44, 1839–1842.
[225] a) W. Zhao, F. Kong, Carbohydr. Res. 2004, 339, 1779–1786;b) W. Zhao, F. Kong, Bioorg. Med. Chem. 2005, 13, 121–130.
[226] W. Zhao, F. Kong, Carbohydr. Res. 2005, 340, 1673–1681.[227] M. Alpe, S. Oscarson, P. Svahnberg, J. Carbohydr. Chem.
2003, 22, 565–477.[228] a) M. Alpe, S. Oscarson, P. Svahnberg, J. Carbohydr. Chem.
2004, 23, 403–416; b) J. Vesely, L. Rydner, S. Oscarson, Car-bohydr. Res. 2008, 343, 2200–2208.
[229] K. Zegelaar-Jaarsveld, S. A. W. Smits, G. A. van der Marel,J. H. van Boom, Bioorg. Med. Chem. 1996, 4, 1819–1832.
[230] S. Oscarson, M. Alpe, P. Svahnberg, A. Nakouzi, A. Casadev-all, Vaccine 2005, 23, 3961–3972.
L. Morelli, L. Poletti, L. LayMICROREVIEW[231] A. Nakouzia, T. Zhanga, S. Oscarson, A. Casadevall, Vaccine
2009, 27, 3513–3518.[232] a) Y. J. Kim, A. Varki, Glycoconjugate J. 1997, 14, 569–576;
b) S. Hakomori, Acta Anat. 1998, 161, 79–90.[233] S. Hakomori, Adv. Cancer Res. 1989, 52, 257–331.[234] G. Ragupathi, R. R. Koganty, D. X. Qiu, K. O. Lloyd, P. O.
Livingston, Glycoconjugate J. 1998, 15, 217–221.[235] a) T. K. Park, I. J. Kim, S. H. Hu, M. T. Bilodeau, J. T. Ran-
dolph, O. Kwon, S. J. Danishefsky, J. Am. Chem. Soc. 1996,118, 11488–11500; b) J. R. Allen, J. G. Allen, X. F. Zhang,L. J. Williams, A. Zatorski, G. Ragupathi, P. O. Livingston,S. J. Danishefsky, Chem. Eur. J. 2000, 6, 1366–1375.
[236] S. J. Danishefsky, M. T. Bilodeau, Angew. Chem. 1996, 108,1482; Angew. Chem. Int. Ed. Engl. 1996, 35, 1380–1419.
[237] G. Ragupathi, T. K. Park, S. L. Zhang, I. J. Kim, L. Graber,S. Adluri, K. O. Lloyd, S. J. Danishefsky, P. O. Livingston,Angew. Chem. 1997, 109, 66; Angew. Chem. Int. Ed. Engl.1997, 36, 125–128.
[238] I. Jeon, K. Iyer, S. J. Danishefsky, J. Org. Chem. 2009, 74,8452–8455.
[239] a) S. J. Danishefsky, V. Behar, J. T. Randolph, K. O. Lloyd, J.Am. Chem. Soc. 1995, 117, 5701–5711; b) V. Kudryashov,H. M. Kim, G. Ragupathi, S. J. Danishefsky, P. O. Livingston,K. O. Lloyd, Cancer Immunol. Immunother. 1998, 45, 281–286; c) P. Sabbatini, V. Kudryashov, G. Ragupathi, S. J. Dani-shefsky, P. O. Livingston, W. Bornmann, M. Spassova, A. Za-torski, D. Spriggs, C. Aghajanian, S. Soignet, M. Peyton, C.O’Flaherty, J. Curtin, K. O. Lloyd, Int. J. Cancer 2000, 87,79–85.
[240] a) S. D. Kuduk, J. B. Schwarz, X. T. Chen, P. W. Glunz, D.Sames, G. Ragupathi, P. O. Livingston, S. J. Danishefsky, J.Am. Chem. Soc. 1998, 120, 12474–12485; b) L. A. Holmberg,B. M. Sandmaier, Expert Rev. Vaccines 2004, 3, 655–663.
[241] a) P. P. Deshpande, H. M. Kim, A. Zatorski, T. K. Park, G.Ragupathi, P. O. Livingstone, D. Live, S. J. Danishefsky, J.Am. Chem. Soc. 1998, 120, 1600–1614; b) G. Ragupathi, P. P.Deshpande, D. M. Coltart, H. M. Kim, L. J. Williams, S. J.Danishefsky, P. O. Livingston, Int. J. Cancer 2002, 99, 207–212; c) T. Buskas, Y. Li, G. J. Boons, Chem. Eur. J. 2005, 11,5457–5467.
[242] a) J. R. Allen, S. J. Danishefsky, J. Am. Chem. Soc. 1999, 121,10875–10882; b) L. M. Krug, G. Ragupathi, C. Hood, M. G.Kris, V. A. Miller, J. R. Allen, S. J. Keding, S. J. Danishefsky,J. Gomez, L. Tyson, B. Pizzo, V. Baez, P. O. Livingston, Clin.Cancer Res. 2004, 10, 6094–6100.
[243] G. Ragupathi, P. Damani, G. Srivastava, O. Srivastava, S. J.Sucheck, Y. Ichikawa, P. O. Livingston, Cancer Immun. Im-munother. 2009, 58, 1397–1405.
[244] A. Franco, Anti-Cancer Agents Med. Chem. 2008, 8, 86–91.[245] a) S. Adluri, F. Helling, S. Ogata, S. Zhang, S. H. Itzkowitz,
K. O. Lloyd, P. O. Livingston, Cancer Immnol. Immunother.1995, 41, 185–192; b) S. Zhang, L. A. Walberg, S. Ogata,S. H. Itzkowitz, R. R. Koganty, M. Reddish, S. S. Ghandi,B. M. Longenecker, K. O. Lloyd, P. O. Livingston, CancerRes. 1995, 55, 3364–3368; c) S. F. Slovin, G. Ragupathi, C.Musselli, K. Olkiewicz, D. Verbel, S. D. Kuduk, J. B.Schwarz, D. Sames, S. J. Danishefsky, P. O. Livingston, H. I.Scher, J. Clin. Oncol. 2003, 21, 4292–4298.
[246] S. J. Danishefsky, J. R. Allen, Angew. Chem. 2000, 112, 882;Angew. Chem. Int. Ed. 2000, 39, 836–863 and references citedtherein.
[247] S. D. Kuduk, J. B. Schwarz, X. T. Chen, P. W. Glunz, D.Sames, G. Ragupathi, P. O. Livingston, S. J. Danishefsky, J.Am. Chem. Soc. 1998, 120, 12474–12485.
[248] F. Slovin, G. Ragupathi, C. Musselli, C. Fernandez, M. Diani,D. Verbel, S. J. Danishefsky, P. O. Livingston, H. I. Scher,Cancer Immunol. Immunother. 2005, 54, 694–702.
[249] J. B. Schwarz, S. D. Kuduk, T. Chen, D. Sames, P. W. Glunz,S. J. Danishefsky, J. Am. Chem. Soc. 1999, 121, 2662–2673.
[250] D. Sames, X. T. Chen, S. J. Danishefsky, Nature 1997, 389,587–591.
[251] a) P. W. Glunz, S. Hintermann, J. B. Schwarz, D. Kuduk,X. T. Chen, L. J. Williams, D. Sames, S. J. Danishefsky, V. Ku-dryashov, K. O. Lloyd, J. Am. Chem. Soc. 1999, 121, 10636–10637; b) P. W. Glunz, S. Hintermann, L. J. Williams, J. B.Schwarz, D. Kuduk, V. Kudryashov, K. O. Lloyd, S. J. Dani-shefsky, J. Am. Chem. Soc. 2000, 122, 7273–72793.
[252] E. Kagan, G. Ragupathi, S. S. Yi, C. A. Reis, J. Gildersleeve,D. Kahne, H. Clausen, S. J. Danishefsky, P. O. Livingston,Cancer Immunol. Immunother. 2005, 54, 424–430.
[253] G. Ragupathi, S. Cappello, S. S. Yi, D. Canter, M. Spassova,W. G. Bornmann, S. J. Danishefsky, P. O. Livingston, Vaccine2002, 20, 1030–1038.
[254] a) L. J. Williams, C. R. Harris, P. W. Glunz, S. J. Danishefsky,Tetrahedron Lett. 2000, 41, 9505–9508; b) J. R. Allen, C. R.Harris, S. J. Danishefsky, J. Am. Chem. Soc. 2001, 123, 1890–1897.
[255] G. Ragupathi, D. M. Coltart, L. J. Williams, F. Koide, E. Ka-gan, J. R. Allen, C. Harris, P. W. Glunz, P. O. Livingston, S. J.Danishefsky, Proc. Natl. Acad. Sci. USA 2002, 99, 13699–13704.
[256] a) S. J. Keding, S. J. Danishefsky, Proc. Natl. Acad. Sci. USA2004, 101, 11937–11942; b) G. Ragupathi, F. Koide, P. O. Liv-ingston, Y. S. Cho, A. Endo, Q. Wan, M. K. Spassova, S. J.Keding, J. R. Allen, O. Ouerfelli, R. M. Wilson, S. J. Danish-efsky, J. Am. Chem. Soc. 2006, 128, 2715–2725; c) J. Zhu,Q. Wan, D. Lee, G. Yang, M. K. Spassova, O. Ouerfelli, G.Ragupathi, P. Damani, P. O. Livingston, S. J. Danishefsky, J.Am. Chem. Soc. 2009, 131, 9298–9303.
[257] J. Zhu, Q. Wan, G. Ragupathi, C. M. George, P. O. Living-ston, S. J. Danishefsky, J. Am. Chem. Soc. 2009, 131, 4151–4158.
[258] D. Lee, S. J. Danishefsky, Tetrahedron Lett. 2009, 50, 2167–2170.
[259] T. Toyokuni, B. Dean, S. P. Chai, D. Boivin, S. Hakomori,A. K. Singhal, J. Am. Chem. Soc. 1994, 116, 395–396.
[260] V. Kudryashov, P. W. Glunz, L. J. Williams, S. Hintermann,S. J. Danishefsky, K. O. Lloyd, Proc. Natl. Acad. Sci. USA2001, 98, 3264–3269.
[261] C. Geraci, G. M. Consoli, E. Galante, E. Bousquet, M. Pap-palardo, A. Spadaro, Bioconjugate Chem. 2008, 19, 751–758.
[262] a) B. A. Cobb, Q. Wang, A. O. Tzianabos, D. L. Kasper, Cell2004, 117, 677–687; b) B. A. Cobb, D. L. Kasper, Cell. Micro-biol. 2005, 7, 1398–1403; c) S. K. Mazmanian, D. L. Kasper,Nat. Rev. Immunol. 2006, 6, 849–858.
[263] R. A. De Silva, Q. Wang, T. Chidley, D. K. Apulage, P. R.Andreana, J. Am. Chem. Soc. 2009, 131, 9622–9623.
[264] S. Gallorini, F. Berti, G. Mancuso, R. Cozzi, M. Tortoli, G.Volpini, J. L. Telford, C. Beninati, D. Maione, A. Wack, Proc.Natl. Acad. Sci. USA 2009, 106, 17481–17486.
[265] a) J. Taylor-Papadimitriou, J. Burchell, D. W. Miles, M. Daz-iel, Biochim. Biophys Acta Mol. Bases Dis. 1999, 1455, 301–313; b) T. Ninkovic, L. Kinarsky, K. Engelmann, V. Pisarev,S. Sherman, O. J. Finn, F. G. Hanisch, Mol. Immunol. 2009,47, 131–140; c) A. L. Sørensen, C. A. Reis, M. A. Tarp, U.Mandel, K. Ramachandran, V. Sankaranarayanan, T. Schien-tek, R. Graham, J. Taylor-Papadimitriou, M. A. Holling-worth, J. Burchel, H. Clausen, Glycobiology 2006, 16, 96–107;d) T. Ju, R. P. Aryal, C. J. Stowell, R. D. Cummings, J. CellBiol. 2008, 182, 531–542.
[266] S. Dziadek, D. Kowalczyk, H. Kunz, Angew. Chem. 2005,117, 7798; Angew. Chem. Int. Ed. 2005, 44, 7624–7630.
[267] S. Dziadek, A. Hobel, E. Schmitt, H. Kunz, Angew. Chem.2005, 117, 7803; Angew. Chem. Int. Ed. 2005, 44, 7630–7635.
[268] U. Westerlind, A. Hobel, N. Gaidzik, E. Schmitt, H. Kunz,Angew. Chem. 2008, 120, 7662; Angew. Chem. Int. Ed. 2008,47, 7551–7556.
Synthetic Oligosaccharide Antigens for Vaccine Formulation
[269] A. Kaiser, N. Gaidzik, U. Westerlind, D. Kowalczyk, A. Ho-bel, E. Schmitt, H. Kunz, Angew. Chem. 2009, 121, 7688; An-gew. Chem. Int. Ed. 2009, 48, 7551–7555.
[270] A. Hoffmann-Röder, A. Kaiser, S. Wagner, N. Gaidzik, D.Kowalczyk, U. Westerlind, B. Gerlitzki, E. Schmitt, H. Kunz,Angew. Chem. Int. Ed. 2010, 49, 8498–8503.
[271] A. Kaiser, N. Gaidzik, T. Becher, C. Menge, K. Groh, H. Cai,Y.-M. Li, B. Gerlitzki, E. Schmitt, H. Kunz, Angew. Chem.Int. Ed. 2010, 49, 3688–3692.
[272] G.-A. Cremer, N. Bureaud, V. Piller, H. Kunz, F. Piller, A. F.Delmas, ChemMedChem 2006, 1, 965–968.
[273] R. Lo-Man, S. Bay, S. Vichier-Guerre, E. Deriaud, D. Canta-cuzene, C. Leclerc, Cancer Res. 1999, 59, 1520–1524.
[274] R. Lo-Man, S. Vichier-Guerre, R. Perraud, E. Deriaud, V.Huteau, L. BenMohamed, O. M. Diop, P. O. Livingston, S.Bay, C. Leclerc, Cancer Res. 2004, 64, 4987–4994.
[275] S. Grigalevicius, S. Chierici, O. Renaudet, R. Lo-Man, E. De-riaud, C. Leclerc, P. Dumy, Bioconjugate Chem. 2005, 16,1149–1159.
[276] I. Jeon, D. Lee, I. J. Krauss, S. J. Danishefsky, J. Am. Chem.Soc. 2009, 131, 14337–14344.
[277] T. Buskas, S. Ingale, G. J. Boons, Angew. Chem. 2005, 117,6139; Angew. Chem. Int. Ed. 2005, 44, 5985–5988.
[278] S. Ingale, M. A. Wolfert, J. Gaekwad, T. Buskas, G.-J. Boons,Nature Chem. Biol. 2007, 3, 663–667.
[279] P. E. Dawson, T. W. Muir, I. Clarklewis, S. B. H. Kent, Sci-ence 1994, 266, 776–779.
[280] S. Ingale, T. Buskas, G.-J. Boons, Org. Lett. 2006, 8, 5785–5788.
[281] S. Ingale, M. A. Wolfert, T. Buskas, G.-J. Boons, ChemBio-Chem 2009, 10, 455–463.
[282] D. R. Bundle, Nature Chem. Biol. 2007, 3, 605–606.[283] a) B. Macher, U. Galili, Biochim. Biophys. Acta Gen. Subj.
Biochim. Biophys. Acta 2008, 1780, 75–88; b) U. M. Abdel-Motal, K. Wiglesworth, U. Galili, Vaccine 2009, 27, 3072–3082.
[284] S. Sarkar, S. A. Lombardo, D. N. Herner, R. S. Talan, K. A.Wall, S. J. Sucheck, J. Am. Chem. Soc. 2010, 132, 17236–17246.
[285] O. Renaudet, L. Ben Mohamed, G. Dasgupta, I. Bettahi, P.Dumy, ChemMedChem 2008, 3, 737–741.
[286] I. Betahi, G. Dasgupta, O. Renaudet, A. A. Chentoufi, X.Zhang, D. Carpenter, S. Yoon, P. Dumy, L. Ben Mohamed,Cancer Immunol. Immunother. 2009, 58, 187–200.
[287] K. L. Mahal, K. J. Yarema, C. R. Bertozzi, Science 1997, 276,1125–1128.
[288] Z. Guo, in: Carbohydrate-Based Vaccines and Immunotherap-ies (Eds.: Z. Guo, G.-J. Boons), John Wiley & Sons, Hoboken,2009, pp. 313–331.
[289] M. Bomsel, D. Tudor, A.-S. Drillet, A. Alfsen, Y. Ganor, M.-G. Roger, N. Mouz, M. Amacker, A. Chalifour, L. Diomede,G. Devillier, Z. Cong, Q. Wei, H. Gao, C. Qin, G.-B. Yang,R. Zurbriggen, L. Lopalco, S. Fleury, Immunity 2011, 34,269–280.
Received: March 2, 2011Published Online: August 1, 2011
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