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able of contents1. Carbohydrate-based vaccines: challenges and opportunities...................................................................... 1

Bibliography...................................................................................................................................................... 24

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Document 1 of 1

Carbohydrate-based vaccines: challenges and opportunitiesAuthor: Huang, Yen-LinProQuest document link

Abstract:Advances in the synthesis of oligo- or polysaccharides and new technologies developed inglycobiology studies have opened a new avenue in carbohydrate vaccine design. In principle, various types of

cell-surface epitopes, characteristic of the invading organism or related to aberrant growth of cells, can be

applied to develop vaccines. Numerous promising carbohydrate-based vaccine candidates have been prepared

in recent years. This article, primarily for general readers, briefly presents the recent advances involving

carbohydrate-based vaccines, including antibacterial, antiparasite, anticancer and antivirus vaccines.

Full text: Figure 1. Current licensed Haemophilus influenzaeserotype b conjugate vaccines. CRM: Diphtheriatoxin mutant; OMPC: Neisseria meningitidisserogroup B outer membrane protein complex; PRP: Poly-ribosyl

ribitol phosphate; TT: Tetanus toxoid. Adapted from [26-30].

(Figure omitted. See article PDF.)

Figure 2. Glycosylphosphatidylinositol-based antimalaria vaccine. GPI: Glycosylphosphatidylinositol; KLH:

Keyhole limpet hemocyanin. Adapted from [32,35].


Figure 3. Synthesis of lipophosphoglycan-based antileishmania vaccine. A)Synthetic antileishmania vaccinewith different phosphoglycan structures conjugated to TetC. B)Synthesis of Leishmania donovanicaptetrasaccharide glycoconjugate with influenza HA. GPI: Glycosylphosphatidylinositol; HA: Hemagglutinin; LPG:

Lipophosphoglycan; TCEP: Tris(2-carboxyethyl)phosphine; TetC: Tetanus toxin fragment C. Adapted from [39-

41].


Figure 4. Monovalent clustered keyhole limpet hemocyanin-conjugated vaccines. (c): Cluster; Gb3:

Globosyltriaoside; KLH: Keyhole limpet hemocyanin; MUC5Ac: Mucin 5, subtypes A and C; Tn: 2-6--N-

acetylgalactosamine. Adapted from [61-64].


Figure 5. Unimolecular pentavalent vaccine conjugate. The construct contains: Globo H, STn, Tn, TF and GM2

antigens conjugated to KLH and pentavalent MUC1 to KLH. Globo H: Globohexaosylceramide; GM:

Gangliosidoses; KLH: Keyhole limpet hemocyanin; MUC: Mucin; STn: Sialyl 2-6-- N-acetylgalactosamine; TF:

Thomsen-Friedenreich; Tn: 2-6--N-acetylgalactosamine. Adapted from [68-70].


Figure 6. Fully synthetic multiple-component vaccines. A)Tn glycopeptides conjugated with Pam3Cys. B)Thetwo-component vaccine contains: Tn-, TF- or STF-modified MUC1 glycopeptide and Pam3CysSK4. C)Thetrimeric branched vaccine contains: unglycosylated Tn, TF-linked MUC1 glycopeptide and a Th epitope,

PADRE. D)The three-component vaccine contains: Tn antigen, Pam3CysSK4 built-in adjuvant and a Thepitope. E)The four-component vaccine contains: Tn-RAFT cyclic peptide, Pam adjuvant, CD8+T-cell epitopeand CD4

+T-cell eitope. MUC: Mucin; OVA: Ovalbumin; PADRE: Pan HLA-DR-binding epitope; Pam: Palmitic

acid; Pam3Cys: Tripalmitoyl-S-glyceryl-cysteinylserine; Pan-DR: CD4+T-helper epitope peptide; RAFT:

Regioselectively addressable functionalized template; SK4: Serine-lysine-lysine-lysine-lysine; STF: Sialyl

Thomsen-Friedenreich; TF: Thomsen-Friedenreich; Th: T helper; TLR: Toll-like receptor; Tn: 2-6--N-acetylgalactosamine. Adapted from [73-78].


Figure 7. Synthetic anti-HIV glycoconjugates. A)GlcNAc2Man

9bivalent glycopeptides OMPC conjugate, B)

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Man9glycodendron BSA conjugate, C)template-assembled of 6-fluorine-modified Man

4conjugate and D)Man

9-Qb bacteriophage conjugate. BSA: Bovine serum albumin; OMPC: Neisseria meningitidisserogroup B outer

membrane protein complex; p:D-proline. Adapted from [84-87].


Beyond their traditionally accepted roles as energy sources and structural polymers, glycans are associated

with cancer metastasis, protein stabilization, pathogen infection and the immune response [1,2]. Thus,

characterization and reconstruction of carbohydrate epitopes with authentic composition and presentation has

become one of the major goals in glycoscience. The attractive carbohydrate-based vaccine targets include

unique glycan structures on the surface of diverse pathogens and the aberrant glycosylation on malignant cells.

Francis and Tillett first reported carbohydrate-based vaccines in 1930 [3]. They found that injection of type-

specific polysaccharides could induce antibodies for heterologous types of pneumococci. In the mid-1940s,

Macleod et al.were the first to use polysaccharide as an immunogen against pneumococcal infection [4].

Bacterial pathogens possess a cell-surface capsular polysaccharide or a lipopolysaccharide shell. These

immense polysaccharides hide the cell-surface component of the bacteria from attack by the host immune

system and prevent antibody-triggered complement activation and phagocytosis. Therefore, antibodies that

specifically target to the bacterial surface polysaccharides may enhance elimination of the pathogens. In 1950,

Heidelberger et al.demonstrated that pneumococcal capsular polysaccharide vaccines could induce high titers

of type-specific antibodies that lasted for 5-8 months [5]. However, this discovery did not arouse much interest

until 1977, when PPV-14, a capsular polysaccharide vaccine derived from 14 pneumococcal serotypes, was

introduced. Later, the same company developed the Pneumovax 23 vaccine, which contains isolated

polysaccharides from 23 out of approximately 90 known serotypes [6,201]. Although a capsular polysaccharide

vaccine is effective in healthy adult populations, it arouses insufficient immunity in infants, young children and

the elderly; populations who are the most vulnerable to bacterial infections due to an immature or compromised

immune system.

Apart from the capsular polysaccharide vaccine, Avery and Goebel first presented the highly immunogenic

pneumococcal glycoconjugate vaccine by coupling carbohydrate to carrier protein [7]; the success of this

method has been widely accepted as a breakthrough in vaccine development. To date, four types of

glycoconjugate vaccines have been licensed that successfully prevent bacterial infections caused by

Haemophilus influenzaetype b, Neisseria meningitidis, Salmonella enterica Serovar Typhiand Streptococcus

pneumoniae[8]. Moreover, several vaccines are undergoing clinical evaluation in Phase II trials against

Staphylococcus aureus, Shigella sonnei, Shigella flexneriand many others.

After the success of antibacterial glycoconjugate vaccines, researchers shifted their attention to the

development of antiprotozoan, antiviral and anticancer vaccines. Advances in carbohydrate chemistry and an

explosion in glycomics have paved the way for vaccine design for a wide variety of diseases. Although manyexcellent reviews have already discussed the specific applications of carbohydrate-based vaccines, we intend

to provide a broader spectrum of recent discoveries in carbohydrate-based vaccination.

Antibacterial vaccinesDespite modern medical advances, bacterial infections remain a major cause of death for infants and children,

particularly in developing countries. Vaccine development could bring such life-threatening diseases to a

minimum. Among many types of antibacterial vaccine, Macleod et al.first successfully created a nonprotein

vaccine with isolated bacterial capsular polysaccharide (CPS) for preventing pneumonia [4]. Bacterial CPSs are

attractive vaccine targets because they are mostly virulence factors, while nonencapsulated bacteria have

reduced pathogenicity. Notable triumphs of polysaccharide vaccines have been made against several bacterial

infections, including S. pneumoniae, H. influenzaetype b, N. meningitidisand S. typhi. However, because

polysaccharides elicit insufficient immune responses, CPS vaccines remain ineffective for infants, the most

vulnerable population to infections.



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Polysaccharides have been considered as T-cell-independent type 2 (TI-2) antigens. In the absence of T-cell-

mediated activation, TI-2 antigens stimulate predominantly low-affinity, non-class-switching IgM antibody

production. Moreover, TI-2 antigens generally do not induce antibody affinity maturation and immunological

memory. To increase the immunogenicity, Avery and Goebel first conjugated oligosaccharides to an

immunogenic carrier protein [7]. The glycoconjugate can be digested by antigen-presenting cells and the

peptide fragments can be preented to the MHC-II complex. Recognition of the peptide-loaded MHC-II complex

and costimulatory signals can activate Th cells and induce Th-2 cytokine production. Th-2-bias cytokines can

further facilitate B-cell maturation into antibody-secreting plasma cells and immunological memory cells. In order

to enhance immunogenicity and efficacy in infants and the elderly, numerous glycoconjugate vaccines have

been investigated and have become widely used for many national immunization programs. In the following

section, we will describe current licensed glycoconjugate vaccines and some promising vaccine candidates that

are undergoing clinical evaluation (Table 1).

Neisseria me ningitidisNeisseria meningitidis, a Gram-negative diplococcal bacterium, causes sporadic bacterial meningitis in

industrialized countries and epidemics in Africa and Asia. The meningococcal disease mostly affects young

adults and children, especially those under 2 years of age. The epidemiology of meningococcal disease

worldwide is complicated by the presence of 12 different serotypes, which are classified according to the

antigenic structure of their polysaccharide capsules. The vast majority of diseases are caused by five

serogroups (A, B, C, Y and W-135) of the bacteria. Group A occurs predominantly in developing countries; while

groups B, C and Y are the major causes of meningitis in developed countries.

The first polysaccharide vaccine used capsular polysaccharides of group A and C N. meningitidisas the

immunogens. The bivalent vaccine was able to induce specific antibody in humans against the respective

serogroups and was subsequently licensed in the USA in 1976 [9]. The current use of a quadrivalent vaccine

(quadrivalent meningococcal polysaccharide vaccine [MPSV4]) against group A, C, W-135 and Y was approved

in 1981. However, the development of quadrivalent polysaccharide vaccines still encounters a variety of

limitations:

* Short protection period and poor immunogenicity in infants under 2 years of age [10];

* Ineffectiveness in attempts to overcome the poor immunogenic properties via repeat immunization [11];

* Deficient protection for group B meningitidis (still the major cause of meningococcal disease worldwide).

Therefore, the vaccine is more suitable for short-term protection, such as for travelers, rather than a national

vaccination campaign. In 2005, a quadrivalent (A, C, Y and W-135) diphtheria conjugate vaccine (MCV-4,

Menactra, Sanofi Pasteur Inc., PA, USA) was developed to improve immunogenicity in young children [12]. In

February 2010, an alternative vaccine (MenACWY-CRM, Menveo, Novartis Vaccines and Diagnostics, Inc.,

MA, USA) was licensed and recommended for individuals aged 11 to 55 years based on its statistically higherseroresponse and comparable safety profile to the older vaccine [13].

However, there is still no effective vaccine against group B meningitidis. The major difficulty in this type of

vaccine development is the structural similarity between normal human tissue and conserved antigenic

structure, (2[arrow right]8)-N-acetylneuraminic acid (polysialic acid [PSA]). Concerns about the high risk of an

autoimmune response limits the progression into of human clinical trials. A preclinical study using N-

propionylated PSA conjugate (NPrPSA-TT) showed elevated bactericidal IgG antibodies in mice without the

detection of autoantibody. A similar result was observed in NPrPSA-PorB (group B meningococcal porin

protein) conjugate vaccinated baboons and monkeys [14,15]. However, the vaccine development has been

suspended because the NPrPSA conjugate failed to induce bactericidal antibodies in adult male volunteers [16].

Although discouraging, this pioneering study ensured the feasibility of modified polysaccharides as an attractive

immunogen to induce functional antibodies. Overall, the capsular polysaccharide still remains an achievable

and challenging target for further investigation in human trials.



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Streptococcus pneumoniaeStreptococcus pneumoniae, a Gram-positive -hemolytic bacterium, has been recognized as a major

contributor to invasive pneumonia, bacteremia, meningitis and noninvasive otitis media. Children under 2 years

of age and adults over 65 years of age are most susceptible to infectious pneumococcal disease. There are

more than 90 types of pneumococci, which are classified by different capsular polysaccharide structures,

prevalence and the extent of drug resistance. The vast majority of diseases are caused by the following

serogroups, in descending order: 14, 6, 19, 18, 9, 23, 4, 1 and 15 in developed countries, but 6, 14, 8, 5, 1, 19,

9, 23, 18, 15 and 17 in developing countries [17,18].

The 14-valent polysaccharide vaccine was first described in 1977 and subsequently replaced by a more

effective 23-valent polysaccharide vaccine (PPV23 [Pneumovax, Merck, PA, USA]) in 1983. Pneumovax used

purified pneumoniae capsular polysaccharide from 23 serotypes, which accounted for 88% of bacteremic

pneumococcal diseases. However, this standard vaccine is ineffective for children under 2 years of age and the

high-risk elderly [19,20]. In 2000, a heptavalent vaccine (PCV7 [ Prevnar, Wyeth, PA, USA]), consisting of

conjugates of diphtheria toxin mutant, CRM197

, and 4, 6B, 9V, 14, 18C, 19F and 23F capsular polysaccharides,

was licensed for routine vaccination and prevention of invasive pneumococcal disease in the USA. The PCV7

vaccine was widely used in the developed world with theoretical coverage of 88.7% in North America, but only

43-67% in Asia and Africa, where nonvaccine serotypes 1 and 5 are highly prevalent [21]. As a result, several

other vaccines are undergoing clinical evaluation in order to increase protection from other prevalent serotypes

[22]. In February 2010, a 13-valent pneumococcal conjugate vaccine (PCV13) with additional serotypes

coverage (1, 3, 5, 6A, 7F and 19A) was approved by the US FDA and expected to offer more protective efficacy

for children aged from 2 to 59 months in most pandemic areas [23]. GlaxoSmithKline's Synflorix(TM),

GlaxoSmithKline Biologicals, Rixensart, Belgium (10-valent pneumococcal conjugate) was licensed in Europe in

2010.

Haemophilus influenzae type b)Haemophilus influenzae, a Gram-negative bacillus, can be recognized as six encapsulated groups (a-f), and

normally inhabits the nasopharyngeal region. However, when infection spreads to the cerebrospinal fluid or

bloodstream, H. influenzaeserotype b (Hib) is the main cause of the severe clinical symptoms of invasive

diseases, such as bacteremia, pneumonia and meningitis. Children under 5 years of age are most susceptible

to invasive disease and account for 80% of all cases. The first-generation vaccine, which was based on a

repeating linear poly-ribosyl ribitol phosphate (PRP), became available in 1985 [24]. However, like other

polysaccharide vaccines, the immune response is highly age-dependent. In infants under 2 years of age,

immunogenicity is uniformly poor and boosters do not elicit anamnestic responses [25].

Taking advantage of polysaccharide-protein coupling, second-generation Hib vaccines (HibTiter, PedvaxHIB

[Merck, PA, USA], ActHib

[Sanofi Pasteur Inc., PA, USA] and Vaxem-Hib

[Chiron, CA, USA]) composed ofdepolymerized PRP linked to various carriers were subsequently licensed in the 1990s (Figure 1) [26-30]. The

glycoconjugate vaccines sufficiently protected infections from Hib. and, thus, are recommended for vaccination

campaigns. However, the cost of available vaccine was among the major obstacles preventing it from becoming

part of the routine immunization program in developing countries [31]. To search for new production

alternatives, Verez-Bencomo et al.proposed a feasible methodology for large-scale manufacture of Hib

polysaccharide fragments. The resulting conjugate vaccine exhibited an excellent safety profile and

immunogenicity and was later approved in Cuba as part of the country's national immunization program in 2004

[30].

The successful large-scale synthesis of ribosylribitol oligomers utilized a key step in polycondensation reaction

with H-phosphonate chemistry. Ribosylribitol oligomers were conjugated to thiolated tetanus toxoid (TT) with an

average final formulation of eight repeating units and 1:2.6 synthetic PRP to TT ratio by weight (Figure 1) [30].

The resulting conjugate vaccine demonstrated a comparable bactericidal antibody response to the commercially



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available vaccine (Vaxem-Hib) in the Phase II trial, involving a total of 1141 infants. Subsequently, several

clinical trials also showed that the vaccine was effective in children with a 99.7% success rate and is now part of

Cuba's national immunization program. This successful study demonstrated a feasible alternative synthetic

strategy that obviates the complexity of vaccine production, including bacteria cultivation, purification and

modifications of CPS. In addition, homogenous compositions of a synthetic PRP minimize batch-to-batch

variation and permit higher quality control during the manufacturing process. Overall, the concept of synthetic

strategy provides a new platform for further development of conjugate vaccines against other infectious

diseases.

Antiparasitic vaccinesParasitic infections are among the most prevalent and severe diseases in human and other mammals. Half of

the world's population, especially those in developing countries, are at risk of the most notorious parasitic

diseases (i.e., malaria, leishmaniasis and trypanosomiasis). Although a wide variety of antiparasitic drugs are

available for treating malaria, reinfection and drug resistance have been major issues, notably in the case of

chloroquine. As a result, producing a widely available vaccine that provides high-level protection for a sustained

period is decisively the ultimate goal. However, to date, there is no commercial vaccine available for human

parasites, regardless of the notable success in several bacterial vaccines. Unlike bacterial vaccines, the major

obstacles for developing antiparasitic vaccines are:

* Complex morphological transitions during the parasites' life cycle, which constantly alters targeted antigens;

* The difficulty of large-scale cultivation, purification and characterization of parasitic surface oligosaccharides

(glycocalyx);

* The elicited antibody-mediated immunity does not sufficiently eradicate intracellular parasites.

MalariaWhile remarkable advances have been made in malaria research, this disease remains a major problem in

tropical areas. Half of the world's population still lives at risk of malarial transmission. Malaria infects more than

5% of the world's population and kills an estimated 1 million people, primarily young children, in Africa annually.

A variety of malaria vaccines are currently undergoing clinical trials. In 2007, the WHO reported that 15 vaccine

candidates against different stages of Plasmodium falciparumhad entered Phase I trials and another ten

vaccines were in Phase II trials [202]. However, the majority of vaccine candidates are peptide vaccines, which

are beyond the scope of this article. Here, we will briefly introduce the novel vaccine target based on

carbohydrate epitopes designed by Seeberger et al.[32].

The malaria parasite P. falciparumshowed particularly low levels of N- and O-linked glycosylation but up to 90%

highly conserved glycosylphosphatidylinositol (GPI) modifications on parasite proteins [33]. Parasite GPIs have

been identified as the prominent toxin that may contribute to malaria pathogenesis and cause symptoms

reminiscent of acute malaria in a mouse model. Therefore, the unique and abundant GPI modificationsrendered it a good target for vaccine development. Both Man

3and Man

4GPI have been observed on P.

falciparum, but their relative ratio varied continuously during maturation [34]. However, only the Man4GPI

anchor is displayed on parasite proteins while Man3GPI can exist as the free glycolipid form on the parasite cell

surface. Based on the nontoxic P. falciparumGPI sequence, Seeberger et al.used automated solid-phase

synthesis of the Man4GPI core structure, (Man1[arrow right]2)-(NH

2-CH

2-CH

2-PO

4)-6-Man(1[arrow right]2)-

Man(1[arrow right]6)-Man(1[arrow right]4)-GlcNH2(1[arrow right]6) myo-inositol- 1,2-cyclic-phosphate, and

conjugated it to maleimide-activated ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) carrier proteins

(Figure 2) [32,35]. Mice immunized with P. falciparumGPI synthetic glycoconjugate vaccine were substantially

protected against Plasmodium berghei-induced cerebral malaria, and mortality was reduced. However, there

was no significant difference in parasitemia levels between vaccination and control groups, suggesting that the

prevention of mortality by anti-GPI antibody does not interfere with the parasite proliferation, but possibly

neutralizes the toxic GPIs.



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Although vaccination proved to be effective in the mouse model, the correlation between anti-GPI antibody

specificity and antitoxic immunity level remains unclear. Therefore, Seeberger et al.generated the synthetic GPI

array (seven GPI derivatives) to analyze anti-GPI antibody binding affinity from healthy subjects in an endemic

malaria region [36]. Their results suggested that the fourth mannose plays a key role in recognition of GPI-

reactive antibodies, but that the phosphate ethanolamine on the third mannose generally had no influence on

binding affinity. Overall, Seeberger's pioneering vaccine study and novel microarray platform using synthetic P.

falciparumGPIs proved the feasibility for preventing severe malaria and the possibility for high-throughput

malaria diagnosis. Further development of the glycoconjugate vaccine is currently undergoing clinical evaluation

by Ancora Pharmaceuticals (Cambridge, MA, USA).

LeishmaniasisLeishmaniasis is caused by intracellular protozoa that survive and replicate in intracellular vacuoles within

macrophages. The Leishmaniagenus has traditionally been differentiated into multiple species that cause

cutaneous, mucosal or visceral diseases [37]. Leishmaniasis is typically transmitted by Phlebotominesandflies

and occurs in 88 countries worldwide with an estimated 2 million cases every year. Cutaneous leishmaniasis

accounts for more than half of the Leishmania diseases and is mainly caused by L. major, L. tropicaand L.

mexicana, while L. donovanigives rise to the most severe visceral leishmaniasis. Although a variety of drugs

were developed for Leishmania treatment many decades ago, the most commonly used antimonials remain

expensive. Moreover, significant side effects and prevalent drug resistance make the development of a

prophylactic vaccine an urgent need. However, the vector-borne diseases are particularly difficult to tackle, as

protection must be effective against their arthropod vectors that enhance the transmission of parasites.

The cell surfaces of Leishmaniaspecies share similar lipophosphoglycan components such as the conserved -

6Galb1-4Man1-PO4- disaccharide repeating unit. The lipophosphoglycans are recognized as an important

virulence factor, crucial for the survival of the parasite in the insect vector, and pivotal for the entry of the

parasite into its mammalian host [38]. As a result, extensive studies were focused on synthesis of this definite

polysaccharide structure for vaccine development [39-41]. As reported by Rogers et al., phosphoglycans of

various structures from L. major, L. mexicanaand L. donovaniwere chemically prepared then conjugated to the

peptide of tetanus toxin (Figure 3A). Furthermore, to evaluate vaccine efficacy, groups of mice were immunized

with different glycoconjugates and then directly challenged by L. mexicana-infected sandfly bite. The majority of

glycoconjugates were unable to protect against fly bite challenge. However L. mexicanaglycoconjugate

conferred significant protection compared with tetanus toxin control. The study demonstrated a 50% reduction in

final lesion size and a 94% reduction in parasite burden [40]. The results revealed that the glycoconjugate

vaccine may induce specific antibody production in a highly species-dependent manner. Overall, the vaccine is

the first demonstration that a synthetic glycoconjugate vaccine can have a direct antiparasitic effect in

leishmaniasis rather than only neutralizing antitoxic effects as reported in malaria models [32].Recently, another glyoconjugate vaccine was developed based on the cap tetrasaccharide of L. donovani

(Figure 3B) [39]. The synthetic cap tetrasaccharide antigen was conjugated to succinimide-activated influenza

hemagglutinin (HA) and then formulated into the virosomal carrier, immunostimulating reconstituted influenza

virosomes. In addition, the virosomal formulations elicited both specific IgM and IgG antibodies in a mouse

model and reacted in vitrowith the natural L. donovani. These findings supported the assertion that the vaccine

candidate was highly immunogenic and had a great potential against leishmaniasis. Finally, with the advance of

more precise carbohydrate structure determination [42] and methods for large-scale synthesis, the development

of antileishmania and other parasitic vaccines has become more promising.

Anticancer vaccines immunotherapeutics)Cancer vaccines are vaccines that either prevent infection with cancer-causing viruses (prophylactic) or prevent

re-emergence or treat existing cancers (therapeutic). They are emerging as a possible treatment for human

malignancy, as prophylactic cancer vaccines against hepatitis B virus-associated liver cancer and human



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papilloma virus-associated cervical cancer were successfully developed and licensed. Besides the prophylactic

vaccines that target cancer-causing viruses, therapeutic cancer vaccines are also being developed (e.g.,

Provenge, Dendreon, WA, USA which has recently been approved to treat advanced prostate cancer

patients). The major development of cancer vaccines focuses mainly on specific protein or peptide antigens;

however, abundantly expressed tumor-associated carbohydrate antigens (TACAs) were also recognized as

specific molecular markers and highly correlated with the various stages of cancer progression [43,44].

Although relatively little is known about the role of surface glycans in malignant cells, evidence has shown that

passively administered or vaccine-induced antibodies against TACAs correlate with improved prognosis [45-47].

Therefore, TACAs have been targeted for cancer vaccine development.

The major challenge of targeting TACAs is their poor immunogenicity and TI feature. Without Th-cell

involvement, TI antigens generally induce low-affinity IgM antibodies. However, antibody-dependent cell-

mediated cytotoxicity (ADCC) is important for anticancer action, and IgM does not stimulate ADCC. Besides, the

antibody levels may constantly decline without arousing immunological memory; thus, repeated vaccinations

are required to maintain antibodies at a level suitable to provide protection. Recent advances have been made

in conjugating TACAs to a variety of carrier proteins (CRM197

, TT, outer membrane protein [OMP], bovine

serum albumin [BSA] and KLH) or peptides (mucin [MUC]1) in order to promote Th-cell activation. However,

despite glycoconjugate vaccines proving successful in treating antibacterial infections, hurdles remain for

cancer vaccine development: first, difficulties of large-scale isolation and purification of homogenous defined

TACAs impede vaccine manufacture; and second, TACAs could be recognized as 'self' antigens during specific

stage development and sporadically express in normal tissues; therefore, antibody production is limited.

Although several elegant reviews have discussed many aspects of cancer vaccines [48-51], we will discuss

recent efforts in cancer vaccine development in the following section. Table 2 lists the status of carbohydrate-

based anticancer vaccines.

Monovalent vaccineThe early generation of carbohydrate-based cancer vaccines was monomeric and consisted of a single antigen

(B-cell epitope) conjugated to an immunogenic carrier protein, in order to enhance the immunogenicity of the

carbohydrate epitope. Helling et al.linked the tumor-associated ganglioside to various carriers [52]. They first

extracted the ganglioside antigen GD3 from bovine brain tissue, and then selectively cleaved it with ozone at

the C4-C5 double bond in the ceramide backbone. After introducing an aldehyde group, the GD3 ganglioside

was coupled to the aminolysyl groups of proteins via reductive amination. Conjugates were constructed with a

great variety of carriers, including KLH, BSA, OMP, multiple antigenic peptide (MAP) and polylysine. Of the

proteins tested for conjugation, KLH showed the strongest ability to induce IgM and IgG antibodies in the

presence of immunological adjuvant, QS-21. In contrast to the GD3 conjugates, GD3 antigen alone induced a

weak IgM response and no IgG response. Their study demonstrated that KLH with QS-21 adjuvant was themost promising immunogenic carrier to enhance the antibody response. Consequently, several ganglioside-

based conjugate vaccines were constructed (e.g., GD2-KLH [53] and GM2-KLH [54]). Among the vaccines,

GM2-KLH showed great promise in clinical evaluation [55].

An alternative approach using synthetic chemistry can also be used to prepare highly complex TACAs.

Danishefsky and coworkers first reported synthesis of the complex Globo H antigen by convergent glycal

assembly strategy to KLH conjugate [56-58]. The Globo H-KLH conjugate was synthesized by reductive

amination after ozone cleavage of allyl glycoside. The Globo H epitope to carrier ratio was further improved

(from 150:1 to 720:1) via a MMCCH linker. In the Phase I trial [57], the conjugate vaccine treatment appeared to

be safe and was capable of inducing a high titer of IgM antibodies that participate in complement-mediated

tumor cell lysis. Overall, evidence of disease stability and the decrease of prostate-specific antigen slope in

treated patients suggested the feasibility of a glycoconjugate vaccine. In addition, a similar synthetic approach

was utilized to synthesize monomeric vaccines (e.g., Fucosyl-GM1-KLH [59] and Ley-KLH conjugate [60])



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against other cancer types.

Since TACAs are likely to be presented in clusters at the surface of cancer cells, single antigens on the

chemical clusters were expected to more closely mimic the architecture of the tumor cell surface. Accordingly,

Danishefsky also developed various KLH-conjugated cluster vaccines (Figure 4; e.g., 2-6-- N-

acetylgalactosamine [Tn](c), Thomsen-Friedenreich [TF](c) [61,62], Sialyl 2-6--N -acetylgalactosamine

[STn](c) [63], and Gb3(c) [64]). In the STn(c) vaccine trial, higher levels of IgM and IgG antibodies were induced

than when using the noncluster STn vaccine, suggesting the significance of clustered antigen presentation.

Polyvalent vaccineWhile the monovalent vaccines produced promising outcome in preclinical and clinical trials, Danishefsky and

Livingston's group has been targeting more TACAs with a particular cancer type in a single vaccine. They

established a hexavalent mixture vaccine consisting of six individual KLH conjugate vaccines: GM2, Globo H,

Ley, MUC1, Tn(c) and TF(c). The conjugate vaccine was co-administered with QS-21 to 30 high-risk prostate

cancer patients in a Phase II clinical trial [65]. The vaccine was well tolerated with no clinical adverse reaction

and all patients showed significant antibody production against at least two TACAs. However, compared with

the previous individual monovalent trials, the antibody titers against TACAs were lower. In addition, a similar

clinical trial was performed for patients with epithelial ovarian, fallopian tube or peritoneal cancer. The

heptavalent vaccine consisted of GM2, Globo H, Ley, Tn-MUC1, Tn(c), STn and TF(c), and was

coadministered with QS-21 [66]. Eight out of nine patients in the final evaluation showed elevated immune

response against at least three antigens and substantial complement-dependent cytotoxicity against MCF-7

breast cancer cells. However, except for anti-MUC1 IgG, the conjugate vaccine induced only IgM antibodies

against other TACAs in those patients.

Obviously, polyvalent antigen vaccines can be constructed by covalently linking different antigens into a single

backbone. Danishefsky's group were the first to synthesize the unimolecular pentavalent vaccine, which

contains five prostate cancer-associated carbohydrate antigens (Globo H, Ley, STn, TF and Tn) as superior

mimics of cell-surface antigens [67]. The antigen-containing amino acid monomers were assembled in a linear

sequential manner, from relatively small Tn to complex Globo H antigen. The glycopeptides were assembled

from fully protected glycosylamino acids with terminal fluorenylmethyl carbamate protecting groups by solution-

phase 9-fluorenylmethyloxycarbonyl-based peptide chemistry. The subsequent conjugation to the carrier protein

was achieved via derivatization of KLH with maleimide, and followed by Michael addition of the thiol handle to

obtain a pentavalent conjugate (ratio: 228:1). The evaluation of the immunogenicity of unimolecular pentavalent

vaccine showed higher immune response against Globo H, TF and STn antigens than the corresponding pooled

monovalent vaccines [68]. However, in both vaccines, no detectable response against Leywas observed,

probably owing to the immunotolerance of its high endogenous expression pattern. Moreover, the immune

reactivity analysis by fluorescence-activated cell sorting suggested that the vaccine-induced antibodies werecapable of recognizing three tumor cell lines. The biological results prompted the same group to develop a

second-generation pentavalent vaccine (Figure 5) [68-70]. In construct, first, the previously used Leyantigen

was replaced by tetrasaccharide GM2, because GM2 antibody levels were correlated with survival in the GM2-

alone vaccine human trial [45]. In particular, an improved conjugation protocol (via preservation of the intact

mercapto group to reduce unnecessary disulfide formation) was developed to obtain a higher epitope to carrier

ratio (505:1). Impressively, the second-generation pentavalent vaccine successfully induced antibodies against

all five TACAs and MCF-7 breast cancer cell lines. Furthermore, an additional immunogenic component, MUC1

random repeat, was introduced to trigger the desired T-cell-mediated immunity.

Overall, the synthesis of complex unimolecular polyvalent vaccine utilized a single bioconjugation reaction

rather than multistep low-yielding conjugations for each TACA; therefore, regulatory validation of each individual

component of vaccine mixture could be simplified. Moreover, compared with a mixture of monomeric vaccines,

the unimolecular polyvalent vaccine used less carrier protein, thus, the epitopic immune suppression was



11/26

minimized.

Although TACA-protein conjugate vaccines are promising and undergoing clinical trials, some limitations

remain. First, the bioconjugation reaction is difficult to control, and the product may have undetermined

ambiguous compositions. The resulting batch-to-batch variations therefore influence the clinical evaluation and

vaccine efficacy. Second, the linkers for carbohydrate and protein conjugation may also be immunogenic,

leading to epitopic suppression [71]. Third, highly immunogenic carrier protein, such as KLH, ineluctably elicits a

strong immune response against the carrier itself. However, the undesired and irrelevant antibody production

may cause suppression of targeted epitopes and even interfere with the vaccine efficacy [72].

Fully synthetic multicomponent vaccineTo overcome these issues, fully synthetic homogeneous vaccines were designed. An early attempt was

described by Toyokuni et al.who covalently linked a dimeric Tn antigen to a Toll-like receptor (TLR) ligand,

tripalmitoyl-S-glyceryl-cysteinylserine (Pam3Cys) derived from Escherichia colilipoproteins (Figure 6A) [73].

Although low titers of specific IgG antibodies were induced, this study demonstrated that a small carbohydrate

antigen could elicit an immune response without the involvement of a carrier protein. Those devoid of IgG

antibody isotype indicated the importance of a Th-epitope in antibody affinity maturation. Later, Kunz et al.

developed a trimeric branched construct containing unglycosylated, Tn, and TF bearing MUC1 glycopeptide

with additional Th epitope, PADRE (Figure 6B) to promote antibody class switch and to target the

heterogeneous tumor surface epitopes. In their study, mouse antiserum were raised towards all three antigens

and also recognized human mammary adenocarcinoma cells [74]. In another attempt, Kaiser et al.efficiently

synthesized a modified MUC1 glycopeptide with Tn, TF or STF antigens and TLR-2 ligand Pam3CysSK4 by

fragment condensation (Figure 6C) [75]. The resulting glycoconjugate can selectively induce antisera when in

combination with complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA).

Impressively, Boons et al.developed a three-component vaccine combining all required elements, a B-cell

epitope (Tn antigen), a built-in adjuvant (Pam3CysSK4) and a Th epitope (mouse MHC II-restricted peptide)

(Figure 6D) [76]. The lipid moiety of the vaccine coordinating with liposomal delivery assisted multivalent

antigen presentation and retention into liposomes; thus, the uptake by antigen-presenting cells was enhanced

and both T- and B-cell activation was triggered. Their study showed that the synthetic three-component vaccine

elicited exceptionally high titers of anti-MUC1 IgG antibodies and could further recognize MCF-7 human breast

cancer cells. When coadministered with QS-21, the vaccine did not elicit a significant increase in IgG1 class, but

a mixed Th1/Th2 response was observed. In addition, low titers of antibodies against Th epitopes indicated that

immune suppression was limited.

Later, Renaudet et al.reported a molecular defined four-component glycolipopeptide (GLP) vaccine PAM-OVA

257-264-PADRE-RAFT (-GalNAc)

4based on oxime/disulfide bond formation [77,78]. The GLP vaccine contained:

a cluster of TACA B-cell epitope; a CD4

+

Th epitope peptide (Pan-DR); a CD8

+

cytotoxic T lymphocyte epitopepeptide; and a palmitic acid built-in adjuvant. In their latest study, they took advantage of the synthetic versatility

of regioselectively addressable functionalized template (RAFT) platform to generate a Tn-clustered

glycolipopeptide vaccine (Figure 6E), which facilitated efficient antigen delivery. Furthermore, the built-in CD4+

and CD8+epitopes assisted in sustaining and priming both humoral and cellular immunity. Instead of using

Pam3CysSK4, which spontaneously forms stable aggregates, they exploited mono-palmitic acid, which has

higher solubility and much easier production and purification procedure under GMP conditions. In the animal

study, the self-adjuvanting GLP vaccine was well tolerated without local or adverse reactions. Moreover, the

vaccine showed promising prophylactic effects in a xenograft model. No tumor developed in any of the

vaccinated mice, and the survival rate was 100% when challenged with murine melanoma MO5 tumor cells

during the monitoring period of 90 days [78].

Overall, the new chemically well-defined synthetic vaccines have many advantages and can be made in a

reproducible manner. The incorporation of minimum relevant elements is required for a desired immune



12/26

response and, hence, limits the immune suppression and any unnecessary immune response against other

antigenic epitopes. Moreover, the covalent incorporation of adjuvant-like TLR ligands may enhance the local

production of cytokines at the adjacent site and facilitate the maturation of relevant immune cells [76].

Furthermore, the CD8+epitope of the four-component vaccine is also crucial in cancer immunity in priming a

cytotoxic T-cell response. However, the mutually synergistic or antagonistic effects of humoral and cytotoxic

responses should be thoroughly considered to ensure vaccine efficacy. Despite the advances in vaccine

development, many obstacles remain: first, the single carbohydrate antigen induces antibody against solely

restricted tumor types or in a small portion of patients. Second, whether this synthetic strategy is applicable for

more complex glycans, such as ganglioside, or large-scale synthesis still needs to be verified. Third, the

therapeutic effect of vaccination in directly eradicating tumors is still limited. Fourth, it is difficult to select

appropriate models to elucidate the vaccine efficacy and bridge the gap between preclinical and practical

human cancer therapy.

Antiviral vaccinesVirus infections cause a great variety of diseases, ranging from the common cold, to influenza, to chronic

hepatitis and life-threatening AIDS. Recent studies have shown that the glycoproteins expressed on the

surfaces of various viruses are highly correlated with their virulence and immune evasion. In contrast to bacteria

or parasites, viruses can take advantage of host glycosylation machinery to construct their own outer-surface

glycans. The host-synthesized glycans are considered to be immune tolerated and, thus, enable viruses to

escape immune surveillance. Moreover, the rapidly mutating virus genome can alter glycosylation sites and

increase the structural diversity of viruses. Despite these challenges, recent advances have been made in

developing carbohydrate-based antiviral vaccines, such as HIV and influenza virus.

HIVHIV is a retrovirus that infects cells of the immune system and causes AIDS worldwide. As the WHO reported in

2009, the number of people living with HIV infection reached an estimated 33.4 million, and the number of

deaths caused by AIDS is approximately 2 million annually. Although important progress has been achieved in

preventing new HIV infections and lowering the mortality rate, the total number of infected is continuously

increasing, especially in Africa and East Asia. Therefore, the development of a prophylactic vaccine still remains

an urgent need.

It is well known that the heavily glycosylated gp120 on the surface of HIV can aggravate immune evasion by

shielding peptide epitopes from immune surveillance and promote infection by interaction with dendritic cells.

Moreover, the conserved dense cluster of oligomannose on gp120 has been recognized as the epitope for the

broadly neutralizing 2G12 antibody. As a result, this relatively unique oligomannose cluster has been targeted

for chemical synthesis in order to elicit 2G12-like antibodies. A combination of crystallographic, glycan array and

modeling studies have shown that Man 9(GlcNAc)2at positions 332, 392 and 339 contributes to the gp120-2G12 interaction with nanomolar affinity [79]. Wang et al.reported that using cholic acid and

D-galactose as

oligomannose cluster scaffolds can increase affinity to monoclonal antibody 2G12 in the range of 21-13 M

[80,81]. However, it remains beyond the affinity between gp120 and 2G12 which was found to be in the

nanomolar range. Recently, Wong's group developed synthetic multivalent Man4 and Man9 glycodendron (3-,

9- and 27-mer) via a copper(I)-catalyzed cycloaddition, and the synthetic glycodendron, especially (Man9)9-

dendron, exhibited promising inhibition ability of both gp120 mAb 2G12 and gp120 DC-SIGN in the nanomolar

range [82]. The results indicated the potential of synthetic glycodendrons to inhibit dendritic cell-mediated HIV

infection. In addition, Calarese et al.reported that Man4 of the D1 arm can inhibit 2G12 binding to gp120 as

efficiently as Man9

(GlcNAc)2

, indicating the potential use of Man4 as a minimum recognition immunogen [83].

Unfortunately, the significantly elicited IgG in rabbits induced by synthetic (Man9)9-dendron or Man4-BSA

conjugates can only bind to glycan structures presented by antigens, but does not cross-react with gp120 [84].

Through disappointing, limited progress, several synthetic strategies continuously performed to yield multivalent



13/26

oligomannose mimetics by displaying on Neisseria meningitidisserogroup B outer membrane protein complex

[85], RAFT (cyclic-decapeptides) [86] and Qb bacteriophage (Figure 7) [87]. Overall, proper optimization of:

glycan spacing, flexibility and immunogenicity are required for synthetic oligomannose to adequately induce

2G12-like neutralizing antibody.

Influenza virusInfluenza virus causes acute viral disease of the respiratory tract and affects millions of people each year.

Among the three virus groups, influenza A is notorious for causing major epidemics and pandemics. The virus

serotypes are named according to two surface glycoproteins, HA and neuraminidase (NA). To date, 16 HA and

nine NA serotypes are described, but three major HAs (H1, H2 and H3) and two NAs (N1 and N2) account for

influenza pandemics in humans. In 1918, the Spanish H1N1 pandemic killed approximately 50 million people

worldwide, in 1957 the Asian influenza H2N2 outbreak killed approximately 2 million people and in 1968 the

Hong Kong influenza pandemic was caused by H3N2 [88]. Both HA and NA glycoproteins recognize host

sialylated receptors with diverse binding specificity. In addition, extensive studies showed that the

carbohydrates on HA and NA may assist in protein folding, virus entry, immune evasion and neurovirulence.

Presently available seasonal influenza vaccines are mainly comprised of purified HA/NA blends and are

effective for many people. However, the immunogenicity is reduced for high-risk populations. Thus, research

focusing on the extensively glycosylated HA of virulent strains is urgently needed. Recently, Wong et al.

discovered that systematic simplification of N-glycans on HA (from complex type HAfgto desialyated complex

type HAds

and from high mannose-type HAhm

to GlcNAc HAmg

) resulted in a successive increase in binding

affinity to 2,3-receptor sialosides [89]. Circular dichroism spectra of different HAs also implied that HA with a

single GlcNAc retained the intact secondary structure compared with the fully glycosylated HA. Moreover, HAmg

antiserum showed stronger neutralization and was much more protective than HAfgvaccination in a lethal-dose

of H5N1 challenge study. Overall, HAmg

is a promising vaccine candidate for influenza because it exposes the

conserved peptide epitopes that are much more immunogenic but were originally hidden by massive glycans. In

addition, the HA with a single GlcNAc showed relaxed specificity but enhanced affinity to 2,3-sialoside and can

be more easily prepared (e.g., via yeast). This strategy paves the way for vaccine design. Together with

successful experience, carbohydrate-based vaccines should facilitate the development of vaccines against viral

infections in the future.

Expert commentaryIn this article, we discuss early efforts and the current state of carbohydrate vaccine research for a variety of

human diseases. The prominent successes in bacterial vaccines provide insights into future vaccinology.

However, considerable issues and challenges remain to be addressed. For instance, identification of varying

parasitic epitopes during the parasite's life cycle and stimulation of both humoral and cell-mediated immunity

are major issues in developing parasitic vaccines. In HIV vaccination, the key challenges are: how to design andconstruct proper presentation of a dense cluster of glycans in order to induce functional neutralizing antibody as

well as to broadly neutralize highly mutated virus strains. Cancer vaccine development encounters similar

difficulties in the aforementioned fields; for example, how to overcome the immunotolerance of TACAs to

generate high levels of tumor-specific antibodies, which can eradicate tumor cells. In addition to humoral

immunity, cytotoxic T lymphocyte response is believed to play a major role in cancer immunotherapy. It is now

accepted that glycopeptides can mediate classical MHC-mediated immune responses. Therefore, cellular

immunity is expected to eliminate tumor cells and may provide an additional opportunity for synthetic

multicomponent vaccines. In recent decades, synthetic chemistry and advanced vaccinology have provided

solutions to common obstacles, such as heterogeneity and poor immunogenicity. Although significant

challenges remain, the rapid growth of glycomics and vaccinology will offer opportunities and accelerate the

development of carbohydrate-based vaccines.

Five-year view27 April 2014 Page 11 of 24 ProQuest


14/26

Although there have been great advances in complicated oligosaccharide synthesis by using programmable

one-pot or automatic solid-phase methods, no automatic machine is currently available in the market. To design

a dream machine for oligosaccharide synthesis is a long-term interest in this field. Carbohydrate-based vaccine

development will benefit from advances in carbohydrate synthesis, development in glycan structures analysis

methods and manipulation through the emerging fields of glycochemistry, glycobiology and immunology.

Carbohydrate recognition is a crucial event in many biological processes, including the development of diseases

such as AIDS, swine influenza and cancer. Thus, understanding and recreating authentic carbohydrate epitope

presentation and composition are important goals in glycoscience and have great value in drugs or vaccines

design. For example, carbohydrate epitopes resulting from virus or aberrant glycosylation of cancer cells

represent attractive targets in designing a carbohydrate-based vaccine. Exploring the carbohydrate epitope in

its natural environment enables us to more directly mimic the natural setting in the context of the vaccine. Now,

glycan array is commonly used as a tool for detecting pathogen-specific antibodies and human cancer for

serodiagonostics. Carbohydrate array technology has tremendous potential for accelerating carbohydrate-

based vaccine development. Employing mass spectrometry and other tools for quality control in carbohydrate-

based vaccines is equally important in this field.

Table 1.

Organism

Saccharidecomponents Carrier Development Licensed Ref.

Haemop

hilus

influenzae(type b)

Type b CPS-

derived 12 mers;

Type b CPS (size

reduced); Type b

CPS (high MW);

Type b CPS

(synthetic

oligosaccharides);

Type b CPS

CRM197

;

OMPs; TT;

TT; TT

Licensed;

Licensed;

Licensed;

Licensed(Cuba);

Licensed

HibTiter, Vaxem-

Hib1990;

PedvaxHIB1990;

ActHib1993;

QuimiHib2004;

Hiberix1998 (EU),

2009 (USA)

[26-30]

Neisseria

meningiti

disA, C,

W135

and Y

Group A, C, Y, W-

135 CPS; Group A,

C, Y, W-135 CPS;

Group A, C, Y, W-

135 CPS

None; DT;

CRM197

Licensed;

Licensed;

Licensed

ACWY Vax1981;

Menactra2005;

Menveo2010

[12,13]

N.

meningiti

dis

(group C)

Group C CPS-

derived oligos; De-

O-Ac CPS-derived

oligos

CRM197

; TTLicensed (UK);

Licensed (UK)

Meningitec,

Menjugate;

NeisVac C1999

[11,12]

Plasmodi

um

falciparu

m

Synthetic GPIsOvalbumin

and KLHPreclinical [32]

Salmon

ella

typhi



15/26

Table 2.

Vi CPS;

Vi CPS;

Vi CPS;

O-acetyl

pectin (Vi

mimics)

None; TT; rEPA;

rEPA

Licensed;

Licensed

(India); Phase

III; Phase I

Typhim Vi

1994; Peda

Typh2001

; ; [90]; [91]

Staphyl

ococcu

s

aureus

Type 5

and 8

CPS;

Type 5

and 8

CPS

rEPA; HSAPhase III;

Phase I

StaphVAX

2004[92]; [93]

Strepto

coccus

pneum

oniae

Target antigen Spacer/other epitope Carrier Cancer type Ref.

Monomeric vaccine Globo H CH2CH

2

KLH, BSA Breast

[56] MMCCH KLH Prostate, breast [57,58]

p-nitrophenyl CRM197

, TTBrea

stFuc-GM1

Ceram

ide-

reducti

ve

aminat

ion

KLH Small-cell lung [59] GD2

Ceram

ide-

lacton

e

KLH Melanoma [53] GD3

Ceram

ide-

reducti

ve

aminat

ion

KLH Melanoma[52,9

6]GM2

Ceram

ide-

reducti

ve

aminat

ion



16/26

KLH Melanoma[54,9

7]GM3

Proteo

liposo

mes

OMPC Melanoma [98] Ley CH2

CH2

KLH Ovarian [60] STnCrotyl

linker

KLH; KLHBreast; Breast, ovarian and

colorectal

[99,1

00];

[101,

102]

PSA, NP-PSA

Reduc

tive

aminat

ion

KLH Small-cell lung [103]Monomeric clustervaccine Tn(c)

MBS KLH, PAMProst

ate[61,62] TF(c)

MBS KLHProst

ate[61] STn(c)

STn(c) crotyl linker-MMCCH KLHBrea

st[63]

Gb3(c)

-

MUC5

Ac

Gb3-norleucine-MUC5Ac-

MBSKLH

Ovari

an[64]

Polyvalentvaccinepooledmonomericvaccines )

GD3, Ley, MUC1 and

MUC2

(GD3)-reductive amination;

(Ley)-CH

2CH

2; (MUC1,

MUC2)-MBS

KLHMelanoma;

Ovarian; Breast[104]

GM2, Globo H, Ley, Tn(c)

and TF(c) MUC1 (32mer)

(GM2 )-reductive amination;

(Ley, Globo H)-MMCCH;

(MUC1, Tn, TF)-MBS

KLH Prostate [65]

GM2, Globo H Ley, Tn(c),

TF(c), STn(c) and MUC1

(GM2)-reductive amination;

(Ley, Globo H)-MMCCH;

(MUC1, Tn, TF, STn)-MBS

KLH

Epithelial ovarian,;

fallopian tube or

peritoneal

[66]



17/26

Unimolecular polyvalentvaccine consists ofmultiantigens onunimolecule)

Globo H, Tn, STn, TF, Ley

and (GM2)

Diami

nopro

pyl-

MBS

KLH

Breast

and

prostat

e

[67-69]Globo H, GM2, Tn, STn and

TF

Diami

nopropyl-

MUC

1-

alani

ne-

MBS

KLH Breast

[70] Multicompone nt vaccineFuco

syl

GM1

FucGM1-

norleucine-MHC II

binding peptide-

MBS

KLH

Small-cell lung [105]

Tn

(two

comp

onent

)

Pam3Cys-

aminobutyl-di-Tn[73]

Ley(two component Pam3Cys-peptide-(Le

y)

3

Ovari

an[106]

Tn, TF

or STF

Pam3CysSK4-ethylene

glycol-MUC1Breast [75]

Tn and TF (three-

component

branched)

Pan-

DR

epitop

e-Lys-

MUC1

-Tn-

Ala-

MUC1

-TF

Breast [74]

Tn(thre

e

comp

onent

)

TLR-2 ligand

(Pam3CysSK4)-

Th epitope-MUC1-

Tn

Breast



18/26

Key issues

* Automatic and programmable one-pot glycan synthesis provides an accessible method to prepare large-scale

carbohydrates for vaccine development.

* Development of carbohydrate-based vaccines against bacterial polysaccharides has made significant

progress, leading to widespread clinical applications.

* Prevention of parasitic infection in tropical areas is particularly important. Despite many ongoing trials, no

effective vaccine is currently available.

* No carbohydrate-based anticancer vaccine is currently approved for clinical use; however, there are many

synthetic cancer vaccines based on tumor-associated carbohydrate antigens undergoing clinical trials, including

Phase III clinical trials.

* Development of a carbohydrate-based antivirus vaccine is more challenging. The similarity between human

and viral glycans can lead to immune responses against self structures.

* The majority of oligosaccharides belonging to T-cell-independent antigens mainly induce IgM antibody

production. Assisted by carrier proteins and adjuvants, high titers of IgG can be induced.

Financial &competing interests disclosure

This study was supported by the Academia Sinica, Taiwan and National Science Council, Taiwan. No. NSC 97-2113-M-001-009-MY2 to Chung-Yi Wu. The authors have no other relevant affiliations or financial involvement

with any organization or entity with a financial interest in or financial conflict with the subject matter or materials

discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

References1 Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology3(2), 97-130 (1993). *

Provides an overview of the biological roles of oligosaccharides.

2 Paulson JC, Blixt O, Collins BE. Sweet spots in functional glycomics. Nat. Chem. Biol.2(5), 238-248 (2006).

3 Francis T, Tillett WS. Cutaneous reactions in pneumonia. The development of antibodies following the

intradermal injection of type-specific polysaccharide. J. Exp. Med.52(4), 573-585 (1930).

4 Macleod CM, Hodges RG, Heidelberger M, Bernhard WG. Prevention of pneumococcal pneumonia by

immunization with specific capsular polysaccharides. J. Exp. Med.82(6), 445-465 (1945).

[76,107] Tn (four component)

TLR-

2

ligan

d

(Pam

3Cys

)-

CD8+

epito

pe

(OVA

257-264

)-

CD4+

epito

pe

(Pan-DR) -

Tn

RAFT Breast



19/26

5 Heidelberger M, Dilapi MM, Siegel M, Walter AW. Persistence of antibodies in human subjects injected with

pneumococcal polysaccharides. J. Immunol.65(5), 535-541 (1950).

6 Robbins JB, Austrian R, Lee CJ et al.Considerations for formulating the second-generation pneumococcal

capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups. J. Infect. Dis.148(6),

1136-1159 (1983).

7 Avery OT, Goebel WF. Chemo-immunological studies on conjugated carbohydrate-proteins: II. Immunological

specificity of synthetic sugar-protein antigens. J. Exp. Med.50(4), 533-550 (1929). ** First demonstration that

carbohydrate conjugated to a carrier protein elevated immunogenicity against carbohydrate antigens.

8 Jones C. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Cienc.

77(2), 293-324 (2005).

9 Meningococcal vaccines. MMWR Morb. Mortal. Wkly Rep.34(18), 255-259 (1985).

10 Reingold AL, Broome CV, Hightower AW et al.Age-specific differences in duration of clinical protection after

vaccination with meningococcal polysaccharide A vaccine. Lancet2(8447), 114-118 (1985).

11 Wilder-Smith A. Meningococcal disease: risk for international travellers and vaccine strategies. Travel Med.

Infect. Dis.6(4), 182-186 (2008).

12 Bilukha OO, Rosenstein N. Prevention and control of meningococcal disease. Recommendations of the

Advisory Committee on Immunization Practices (ACIP). MMWR Recomm. Rep.54(RR-7), 1-21 (2005).

13 Licensure of a meningococcal conjugate vaccine (Menveo) and guidance for use - Advisory Committee on

Immunization Practices (ACIP), 2010. MMWR Morb. Mortal. Wkly Rep.59(9), 273 (2010).

14 Jennings HJ, Roy R, Gamian A. Induction of meningococcal group B polysaccharide-specific IgG antibodies

in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J. Immunol.137(5),

1708-1713 (1986).

15 Fusco PC, Michon F, Tai JY, Blake MS. Preclinical evaluation of a novel group B meningococcal conjugate

vaccine that elicits bactericidal activity in both mice and nonhuman primates. J. Infect. Dis.175(2), 364-372

(1997).

16 Bruge J, Bouveret-Le Cam N, Danve B, Rougon G, Schulz D. Clinical evaluation of a group B

meningococcal N-propionylated polysaccharide conjugate vaccine in adult, male volunteers. Vaccine22(9-10),

1087-1096 (2004).

17 Sniadack DH, Schwartz B, Lipman H et al.Potential interventions for the prevention of childhood pneumonia:

geographic and temporal differences in serotype and serogroup distribution of sterile site pneumococcal isolates

from children - implications for vaccine strategies. Pediatr. Infect. Dis. J.14(6), 503-510 (1995).

18 Kirkwood BR, Gove S, Rogers S, Lob-Levyt J, Arthur P, Campbell H. Potential interventions for the

prevention of childhood pneumonia in developing countries: a systematic review. Bull. World Health Organ.

73(6), 793-798 (1995).19 Shapiro ED, Berg AT, Austrian R et al.The protective efficacy of polyvalent pneumococcal polysaccharide

vaccine. N. Engl. J. Med.325(21), 1453-1460 (1991).

20 Melegaro A, Edmunds WJ. The 23-valent pneumococcal polysaccharide vaccine. Part I. Efficacy of PPV in

the elderly: a comparison of meta-analyses. Eur. J. Epidemiol.19(4), 353-363 (2004).

21 Oosterhuis-Kafeja F, Beutels P, Van Damme P. Immunogenicity, efficacy, safety and effectiveness of

pneumococcal conjugate vaccines (1998-2006). Vaccine25(12), 2194-2212 (2007).

22 Wuorimaa T, Kayhty H. Current state of pneumococcal vaccines. Scand J. Immunol.56(2), 111-129 (2002).

23 Licensure of a 13-valent pneumococcal conjugate vaccine (PCV13) and recommendations for use among

children - Advisory Committee on Immunization Practices (ACIP), 2010. MMWR Morb. Mortal. Wkly Rep.59(9),

258-261 (2010).

24 Atkinson W, Hamborsky J, McIntyre L, Wolfe S (Eds). Epidemiology and Prevention of Vaccine-Preventable

Diseases (9th edition). Centers for Disease Control and Prevention, GA, USA (2006).



20/26

25 Kayhty H, Karanko V, Peltola H, Makela PH. Serum antibodies after vaccination with Haemophilus

influenzaetype b capsular polysaccharide and responses to reimmunization: no evidence of immunologic

tolerance or memory. Pediatrics74(5), 857-865 (1984).

26 Guo ZW, Boons GJ. Chapter 2: preparation of glycoconjugate vaccines. In: Carbohydrate based Vaccines

and Immunotherapies.Jogn A (Ed.). Wiley &Sons, Inc., NJ, USA, 55-88 (2009).

27 Anderson PW, Pichichero ME, Insel RA, Betts R, Eby R, Smith DH. Vaccines consisting of periodate-cleaved

oligosaccharides from the capsule of Haemophilus influenzaetype b coupled to a protein carrier: structural and

temporal requirements for priming in the human infant. J. Immunol.137(4), 1181-1186 (1986).

28 Schneerson R, Barrera O, Sutton A, Robbins JB. Preparation, characterization, and immunogenicity of

Haemophilus influenzaetype b polysaccharide-protein conjugates. J. Exp. Med.152(2), 361-376 (1980).

29 Marburg S, Jorn D, Tolman RL et al.Bimolecular chemistry of macromolecules - synthesis of bacterial

polysaccharide conjugates with Neisseria meningitidismembrane-protein. J. Am. Chem. Soc.108(17), 5282-

5287 (1986).

30 Verez-Bencomo V, Fernandez-Santana V, Hardy E et al.A synthetic conjugate polysaccharide vaccine

against Haemophilus influenzaetype b. Science305(5683), 522-525 (2004). * Describes the first fully synthetic

polysaccharide conjugate vaccine available in a routine immunization program.

31 Torano G, Toledo ME, Baly A et al.Phase I clinical evaluation of a synthetic oligosaccharide-protein

conjugate vaccine against Haemophilus influenzaetype b in human adult volunteers. Clin. Vaccine Immunol.

13(9), 1052-1056 (2006).

32 Schofield L, Hewitt MC, Evans K, Siomos MA, Seeberger PH. Synthetic GPI as a candidate anti-toxic

vaccine in a model of malaria. Nature418(6899), 785-789 (2002). * Describes the rational design and chemical

synthesis of a hlycophosphatidylinositol-based glycoconjugate vaccine against malaria.

33 Gerold P, Schofield L, Blackman MJ, Holder AA, Schwarz RT. Structural analysis of the glycosyl-

phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum.

Mol. Biochem. Parasitol.75(2), 131-143 (1996).

34 Schmidt A, Schwarz RT, Gerold P. Plasmodium falciparum: asexual erythrocytic stages synthesize two

structurally distinct free and protein-bound glycosylphosphatidylinositols in a maturation-dependent manner.

Exp. Parasitol.88(2), 95-102 (1998).

35 Hewitt MC, Snyder DA, Seeberger PH. Rapid synthesis of a glycosylphosphatidylinositol-based malaria

vaccine using automated solid-phase oligosaccharide synthesis. J. Am. Chem. Soc.124(45), 13434-13436

(2002).

36 Kamena F, Tamborrini M, Liu X et al.Synthetic GPI array to study antitoxic malaria response. Nat. Chem.

Biol.4(4), 238-240 (2008).

37 Desjeux P. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis.27(5), 305-318 (2004).

38 Mahoney AB, Sacks DL, Saraiva E, Modi G, Turco SJ. Intra-species and stage-specific polymorphisms in

lipophosphoglycan structure control Leishmania donovani-sand fly interactions. Biochemistry38(31), 9813-

9823 (1999).

39 Liu X, Siegrist S, Amacker M, Zurbriggen R, Pluschke G, Seeberger PH. Enhancement of the

immunogenicity of synthetic carbohydrates by conjugation to virosomes: a leishmaniasis vaccine candidate.

ACS Chem. Biol.1(3), 161-164 (2006).

40 Rogers ME, Sizova OV, Ferguson MA, Nikolaev AV, Bates PA. Synthetic glycovaccine protects against the

bite of leishmania-infected sand flies. J. Infect. Dis.194(4), 512-518 (2006).

41 Routier FH, Nikolaev AV, Ferguson MA. The preparation of neoglycoconjugates containing inter-saccharide

phosphodiester linkages as potential anti-Leishmania vaccines. Glycoconj. J.16(12), 773-780 (1999).

42 Mendonca-Previato L, Todeschini AR, Heise N, Previato JO. Protozoan parasite-specific carbohydrate



21/26

structures. Curr. Opin. Struct. Biol.15(5), 499-505 (2005).

43 Ragupathi G. Carbohydrate antigens as targets for active specific immunotherapy. Cancer Immunol.

Immunother.43(3), 152-157 (1996).

44 Hakomori S. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of

anti-cancer vaccines.Adv. Exp. Med. Biol.491, 369-402 (2001).

45 Livingston PO, Wong GY, Adluri S et al.Improved survival in stage III melanoma patients with GM2

antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside. J. Clin. Oncol.12(5), 1036-1044

(1994).

46 Jones PC, Sze LL, Liu PY, Morton DL, Irie RF. Prolonged survival for melanoma patients with elevated IgM

antibody to oncofetal antigen. J. Natl Cancer Inst.66(2), 249-254 (1981).

47 Schindlbeck C, Jeschke U, Schulze Set al.Prognostic impact of Thomsen-Friedenreich tumor antigen and

disseminated tumor cells in the bone marrow of breast cancer patients. Breast Cancer Res. Treat.101(1), 17-25

(2007).

48 Buskas T, Thompson P, Boons GJ. Immunotherapy for cancer: synthetic carbohydrate-based vaccines.

Chem. Commun.36, 5335-5349 (2009).

49 Guo Z, Wang Q. Recent development in carbohydrate-based cancer vaccines. Curr. Opin. Chem. Biol.13(5-

6), 608-617 (2009).

50 Zhu J, Warren JD, Danishefsky SJ. Synthetic carbohydrate-based anticancer vaccines: the Memorial Sloan-

Kettering experience. Expert Rev. Vaccines8(10), 1399-1413 (2009).

51 Astronomo RD, Burton DR. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat.

Rev. Drug Discov.9(4), 308-324 (2010). ** Comprehensive review on the recent development of synthetic

carbohydrate vaccines against a diverse range of pathogens and cancers.

52 Helling F, Shang A, Calves M et al.GD3 vaccines for melanoma: superior immunogenicity of keyhole limpet

hemocyanin conjugate vaccines. Cancer Res.54(1), 197-203 (1994). * Compares the immunogenicity of the

diverse range of carrier proteins used in the ganglioside vaccine for the immunotherapeutic treatment of human

cancers.

53 Zhang H, Zhang S, Cheung NK, Ragupathi G, Livingston PO. Antibodies against GD2 ganglioside can

eradicate syngeneic cancer micrometastases. Cancer Res.58(13), 2844-2849 (1998).

54 Helling F, Zhang S, Shang A et al.GM2-KLH conjugate vaccine: increased immunogenicity in melanoma

patients after administration with immunological adjuvant QS-21. Cancer Res.55(13), 2783-2788 (1995).

55 Slovin SF, Keding SJ, Ragupathi G. Carbohydrate vaccines as immunotherapy for cancer. Immunol. Cell

Biol.83(4), 418-428 (2005).

56 Ragupathi G, Park TK, Zhang SL et al.Immunization of mice with a fully synthetic globo H antigen results in

antibodies against human cancer cells: a combined chemical-immunological approach to the fashioning of ananticancer vaccine.Angew Chem. Int. Ed. Engl.36(1-2), 125-128 (1997).

57 Slovin SF, Ragupathi G, Adluri S et al.Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic

globo H hexasaccharide conjugate in man. Proc. Natl Acad. Sci. USA96(10), 5710-5715 (1999).

58 Gilewski T, Ragupathi G, Bhuta S et al.Immunization of metastatic breast cancer patients with a fully

synthetic globo H conjugate: a Phase I trial. Proc. Natl Acad. Sci. USA98(6), 3270-3275 (2001).

59 Dickler MN, Ragupathi G, Liu NX et al.Immunogenicity of a fucosyl-GM1-keyhole limpet hemocyanin

conjugate vaccine in patients with small cell lung cancer. Clin. Cancer Res.5(10), 2773-2779 (1999).

60 Sabbatini PJ, Kudryashov V, Ragupathi G et al.Immunization of ovarian cancer patients with a synthetic

Lewis(y)-protein conjugate vaccine: a Phase 1 trial. Int. J. Cancer87(1), 79-85 (2000).

61 Kuduk SD, Schwarz JB, Chen XT et al.Synthetic and immunological studies on clustered modes of mucin-

related Tn and TF O-linked antigens: the preparation of a glycopeptide-based vaccine for clinical trials against

prostate cancer. J. Am. Chem. Soc.120(48), 12474-12485 (1998).



22/26

62 Slovin SF, Ragupathi G, Musselli C et al.Fully synthetic carbohydrate-based vaccines in biochemically

relapsed prostate cancer: clinical trial results with -N- acetylgalactosamine-O-serine/threonine conjugate

vaccine. J. Clin. Oncol.21(23), 4292-4298 (2003).

63 Gilewski TA, Ragupathi G, Dickler M et al.Immunization of high-risk breast cancer patients with clustered

STn-KLH conjugate plus the immunologic adjuvant QS-21. Clin. Cancer Res.13(10), 2977-2985 (2007).

64 Zhu J, Wan Q, Ragupathi G, George CM, Livingston PO, Danishefsky SJ. Biologics through chemistry: total

synthesis of a proposed dual-acting vaccine targeting ovarian cancer by orchestration of oligosaccharide and

polypeptide domains. J. Am. Chem. Soc.131(11), 4151-4158 (2009).

65 Slovin SF, Ragupathi G, Fernandez C et al.A polyvalent vaccine for high-risk prostate patients: "are more

antigens better?". Cancer Immunol. Immunother.56(12), 1921-1930 (2007).

66 Sabbatini PJ, Ragupathi G, Hood C et al.Pilot study of a heptavalent vaccine-keyhole limpet hemocyanin

conjugate plus QS21 in patients with epithelial ovarian, fallopian tube, or peritoneal cancer. Clin. Cancer Res.

13(14), 4170-4177 (2007).

67 Keding SJ, Danishefsky SJ. Prospects for total synthesis: a vision for a totally synthetic vaccine targeting

epithelial tumors. Proc. Natl Acad. Sci. USA101(33), 11937-11942 (2004). * Describes the design and chemical

synthesis of the first unimolecular multivalent glycoconjugate vaccine.

68 Ragupathi G, Koide F, Livingston PO et al.Preparation and evaluation of unimolecular pentavalent and

hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine

candidates. J. Am. Chem. Soc.128(8), 2715-2725 (2006).

69 Zhu J, Wan Q, Lee D et al.From synthesis to biologics: preclinical data on a chemistry derived anticancer

vaccine. J. Am. Chem. Soc.131(26), 9298-9303 (2009).

70 Lee D, Danishefsky SJ. 'Biologic' level structures through chemistry: a total synthesis of a unimolecular

pentavalent MUCI glycopeptide construct. Tetrahedron Lett.50(19), 2167-2170 (2009).

71 Buskas T, Li Y, Boons GJ. The immunogenicity of the tumor-associated antigen Lewis(y) may be

suppressed by a bifunctional cross-linker required for coupling to a carrier protein. Chem. Eur. J.10(14), 3517-

3524 (2004).

72 Schutze MP, Leclerc C, Jolivet M, Audibert F, Chedid L. Carrier-induced epitopic suppression, a major issue

for future synthetic vaccines. J. Immunol.135(4), 2319-2322 (1985).

73 Toyokuni T, Dean B, Cai SP, Boivin D, Hakomori S, Singhal AK. Synthetic vaccines - synthesis of a dimeric

Tn antigen-lipopeptide conjugate that elicits immune-responses against Tn-expressing glycoproteins. J. Am.

Chem. Soc.116(1), 395-396 (1994).

74 Cremer GA, Bureaud N, Piller V, Kunz H, Piller F, Delmas AF. Synthesis and biological evaluation of a

multiantigenic Tn/TF-containing glycopeptide mimic of the tumor-related MUC1 glycoprotein. Chem. Med.

Chem.1(9), 965-968 (2006).75 Kaiser A, Gaidzik N, Becker T et al.Fully synthetic vaccines consisting of tumor-associated MUC1

glycopeptides and a lipopeptide ligand of the Toll-like receptor 2. Angew Chem. Int. Ed. Engl.49(21), 3688-

3692 (2010).

76 Ingale S, Wolfert MA, Gaekwad J, Buskas T, Boons GJ. Robust immune responses elicited by a fully

synthetic three-component vaccine. Nat. Chem. Biol.3(10), 663-667 (2007). * Provides insight into the design

and synthesis of a three-component carbohydrate vaccine containing a built-in adjuvant, Th epitope and B-cell

epitope.

77 Renaudet O, BenMohamed L, Dasgupta G, Bettahi I, Dumy P. Towards a self-adjuvanting multivalent B and

T cell epitope containing synthetic glycolipopeptide cancer vaccine. Chem. Med. Chem.3(5), 737-741 (2008).

78 Bettahi I, Dasgupta G, Renaudet O et al.Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine

bearing B cell, CD4+and CD8

+T cell epitopes. Cancer Immunol. Immunother.58(2), 187-200 (2009).

79 Calarese DA, Scanlan CN, Zwick MB et al.Antibody domain exchange is an immunological solution to



23/26

carbohydrate cluster recognition. Science300(5628), 2065-2071 (2003).

80 Wang LX, Ni J, Singh S, Li H. Binding of high-mannose-type oligosaccharides and synthetic oligomannose

clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem. Biol.11(1), 127-134 (2004).

81 Li H, Wang LX. Design and synthesis of a template-assembled oligomannose cluster as an epitope mimic for

human HIV-neutralizing antibody 2G12. Org. Biomol. Chem.2(4), 483-488 (2004).

82 Wang SK, Liang PH, Astronomo RD et al.Targeting the carbohydrates on HIV-1: interaction of

oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl Acad. Sci. USA

105(10), 3690-3695 (2008).

83 Calarese DA, Lee HK, Huang CY et al.Dissection of the carbohydrate specificity of the broadly neutralizing

anti-HIV-1 antibody 2G12. Proc. Natl Acad. Sci. USA102(38), 13372-13377 (2005).

84 Astronomo RD, Lee HK, Scanlan CN et al.A glycoconjugate antigen based on the recognition motif of a

broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies

unable to bind to the self glycans of gp120. J. Virol.82(13), 6359-6368 (2008).

85 Krauss IJ, Joyce JG, Finnefrock AC et al.Fully synthetic carbohydrate HIV antigens designed on the logic of

the 2G12 antibody. J. Am. Chem. Soc.129(36), 11042-11044 (2007).

86 Wang J, Li H, Zou G, Wang LX. Novel template-assembled oligosaccharide clusters as epitope mimics for

HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study. Org. Biomol. Chem.5(10),

1529-1540 (2007).

87 Astronomo RD, Kaltgrad E, Udit AK et al.Defining criteria for oligomannose immunogens for HIV using

icosahedral virus capsid scaffolds. Chem. Biol.17(4), 357-370 (2010).

88 Stevens J, Blixt O, Paulson JC, Wilson IA. Glycan microarray technologies: tools to survey host specificity of

influenza viruses. Nat. Rev. Microbiol.4(11), 857-864 (2006).

89 Wang CC, Chen JR, Tseng YC et al.Glycans on influenza hemagglutinin affect receptor binding and

immune response. Proc. Natl Acad. Sci. USA106(43), 18137-18142 (2009).

90 Lin FY, Ho VA, Khiem HB et al.The efficacy of a Salmonella typhiVi conjugate vaccine in two-to-five-year-

old children. N. Engl. J. Med.344(17), 1263-1269 (2001).

91 Szu SC, Bystricky S, Hinojosa-Ahumada M, Egan W, Robbins JB. Synthesis and some immunologic

properties of an O-acetyl pectin [poly(1[arrow right]4)--D-GalpA]-protein conjugate as a vaccine for typhoid

fever. Infect. Immun.62(12), 5545-5549 (1994).

92 Fattom AI, Horwith G, Fuller S, Propst M, Naso R. Development of StaphVAX, a polysaccharide conjugate

vaccine against S. aureusinfection: from the lab bench to Phase III clinical trials. Vaccine22(7), 880-887

(2004).

93 Tollersrud T, Zernichow L, Andersen SR, Kenny K, Lund A. Staphylococcus aureuscapsular polysaccharide

type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle. Vaccine19(28-29), 3896-3903(2001).

94 Schuerman L, Prymula R, Henckaerts I, Poolman J. ELISA IgG concentrations and opsonophagocytic

activity following pneumococcal protein D conjugate vaccination and relationship to efficacy against acute otitis

media. Vaccine25(11), 1962-1968 (2007).

95 Prymula R, Peeters P, Chrobok V et al.Pneumococcal capsular polysaccharides conjugated to protein D for

prevention of acute otitis media caused by both Streptococcus pneumoniaeand non-typable Haemophilus

influenzae: a randomised double-blind efficacy study. Lancet367(9512), 740-748 (2006).

96 Chapman PB, Wu D, Ragupathi G et al.Sequential immunization of melanoma patients with GD3

ganglioside vaccine and anti-idiotypic monoclonal antibody that mimics GD3 ganglioside. Clin. Cancer Res.

10(14), 4717-4723 (2004).

97 Livingston PO, Adluri S, Helling F et al.Phase 1 trial of immunological adjuvant QS-21 with a GM2

ganglioside-keyhole limpet haemocyanin conjugate vaccine in patients with malignant melanoma. Vaccine



24/26

12(14), 1275-1280 (1994).

98 Guthmann MD, Bitton RJ, Carnero AJ et al.Active specific immunotherapy of melanoma with a GM3

ganglioside-based vaccine: a report on safety and immunogenicity. J. Immunother.27(6), 442-451 (2004).

99 Longenecker BM, Reddish M, Koganty R, MacLean GD. Immune responses of mice and human breast

cancer patients following immunization with synthetic sialyl-Tn conjugated to KLH plus detox adjuvant.Ann. NY

Acad. Sci.690, 276-291 (1993).

100 MacLean GD, Reddish M, Koganty RR et al.Immunization of breast cancer patients using a synthetic

sialyl-Tn glycoconjugate plus detox adjuvant. Cancer Immunol. Immunother.36(4), 215-222 (1993).

101 MacLean GD, Miles DW, Rubens RD, Reddish MA, Longenecker BM. Enhancing the effect of

THERATOPE STn-KLH cancer vaccine in patients with metastatic breast cancer by pretreatment with low-dose

intravenous cyclophosphamide. J. Immunother. Emphasis Tumor Immunol.19(4), 309-316 (1996).

102 MacLean GD, Reddish MA, Koganty RR, Longenecker BM. Antibodies against mucin-associated sialyl-Tn

epitopes correlate with survival of metastatic adenocarcinoma patients undergoing active specific

immunotherapy with synthetic STn vaccine. J. Immunother. Emphasis Tumor Immunol.19(1), 59-68 (1996).

103 Krug LM, Ragupathi G, Ng KK et al.Vaccination of small cell lung cancer patients with polysialic acid or N-

propionylated polysialic acid conjugated to keyhole limpet hemocyanin. Clin. Cancer Res.10(3), 916-923

(2004).

104 Ragupathi G, Cappello S, Yi SS et al.Comparison of antibody titers after immunization with monovalent or

tetravalent KLH conjugate vaccines. Vaccine20(7-8), 1030-1038 (2002).

105 Nagorny P, Kim WH, Wan Q, Lee D, Danishefsky SJ. On the emerging role of chemistry in the fashioning of

biologics: synthesis of a bidomainal fucosyl gm1-based vaccine for the treatment of small cell lung cancer. J.

Org. Chem.74(15), 5157-5162 (2009).

106 Kudryashov V, Glunz PW, Williams LJ, Hintermann S, Danishefsky SJ, Lloyd KO. Toward optimized

carbohydrate-based anticancer vaccines: epitope clustering, carrier structure, and adjuvant all influence

antibody responses to Lewis(y) conjugates in mice. Proc. Natl Acad. Sci. USA98(6), 3264-3269 (2001).

107 Buskas T, Ingale S, Boons GJ. Towards a fully synthetic carbohydrate-based anticancer vaccine: synthesis

and immunological evaluation of a lipidated glycopeptide containing the tumor-associated tn antigen.Angew

Chem. Int. Ed. Engl.44(37), 5985-5988 (2005).

201 Merck &Co. I. Pneumovax 23 (pneumococcal vaccine polyvalent)

www.merck.com/product/usa/pi_circulars/p/pneumovax_23/pneumovax_pi.pdf

202 The Jordan report. Accelerated the development of vaccines 2007

www3.niaid.nih.gov/about/organization/dmid/PDF/Jordan2007.pdf

AuthorAffiliationYen-Lin Huang,

1

Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang,Taipei, 115, Taiwan. [email protected].

MeSH: Bacterial Vaccines -- chemistry, Cancer Vaccines -- chemistry, Humans, Protozoan Vaccines --chemistry, Viral Vaccines -- chemistry, Bacterial Vaccines -- immunology (major), Cancer Vaccines --

immunology (major), Carbohydrates -- immunology (major), Epitopes -- immunology (major), Protozoan

Vaccines -- immunology (major), Viral Vaccines -- immunology (major)

Substance: Bacterial Vaccines; Cancer Vaccines; Carbohydrates; Epitopes; Protozoan Vaccines; ViralVaccines;

Publication title: Expert Review of VaccinesVolume: 9



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Issue: 11

Pages: 1257-74

Publication year: 2010

Publication date: Nov 2010

Year: 2010Publisher: Informa Healthcare, Expert Reviews Ltd.

Place of publication: London

Country of publication: United Kingdom

Publication subject: Medical Sciences--Allergology And Immunology

ISSN: 1476-0584

Source type: Scholarly Journals

Language of publication: EnglishDocument type: Review, Journal Article

DOI: http://dx.doi.org/10.1586/erv.10.120

Accession number: 21087106

ProQuest document ID: 8