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New trends in carbohydrate-based vaccines
Vol. 1, No. 3 2004
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Lead optimization
New trends in carbohydrate-based vaccines Rene´ Roy Department of Chemistry, Universite´ du Que´bec a` Montre´al, P.O. Box 8888, Succ. Centre-Ville, Montre´al, Que´bec, Canada H3C 3P8
Carbohydrate-based vaccines constitute successful therapeutic arsenals against microbial infections, particularly since the advent of bacterial capsular polysaccharide (CPS) protein conjugates. These prophylactic approaches are increasingly more accessible for other therapies such as viral, parasitic, fungal infections and even against certain forms of cancer. Three major discoveries are responsible for rapid growth in this field. In the first instance, the recent commercialization of the first semi-synthetic vaccine against bacterial infections caused by Haemophilus influenza type b, second by the rapid access to complex saccharide structures through one-pot, solution-phase syntheses, and third by automated solid-phase synthesis.
Introduction Bacterial, parasitic and viral infections constitute one of the major health problems worldwide. Drug resistance is rapidly escalating and new drug developments, although faster than in the past with the advent of combinatorial methods, is becoming prohibitive. Therefore, the development of new prophylactic and potent therapeutic vaccines to combat these types of infections is of utmost importance, especially in developing countries. The cell-surface of most pathogens and cancer cells express several structurally variable and sometime cryptic carbohydrate molecules that are thus E-mail address: [email protected]. 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.10.005
Section Editor: Constant A.A. van Boeckel – Organon NV, Oss, The Netherlands The capsular polysaccharides of various pathogenic bacteria have been used for many decades as vaccines. Later it turned out that such vaccines are more efficacious, in particular in childhood, if they are applied as conjugates with non-endogenous proteins. During the past decade, several synthetic methodologies have been developed to prepare well-defined carbohydrate epitopes, which are much smaller than the native polysaccharides and which can be hooked via synthetic linkers to the desired protein. Moreover, peptide synthesis allows the preparation of tailor-made peptide fragments comprising so-called Tcell epitopes. By using various carbohydrate, peptide and conjugation technologies, fully synthetic vaccines can be developed. Such technologies open new avenues not only for bacterial and viral vaccines but also for novel anti-cancer vaccines. Rene´ Roy has long-standing experience in carbohydrate chemistry and conjugation of complex synthetic epitopes to produce synthetic clusters and vaccines. He is one of the contributors towards the first semi-synthetic vaccine against Haemophilus influenza type b that is efficacious in human beings.
exposed at the forefront battle against the immune system. Hence, by triggering a specific and a high-affinity immune response against a particular cell surface carbohydrate antigen, protective and long lasting immunity is thus achieved. This strategy has been very successful with bacterial capsular polysaccharides (CPS) and several such glycoconjugates are now commercially available against Neisseria meningitidis groups A, C, W-135 and Y, Steptococcus pneumoniae (23 serotypes), Haemophilus influenza type b (Hib, see Glossary) and Salmonella typhi [1–5]. For instance, with the advent in the 1990s of protein conjugate vaccines against the Gram-negative organisms Hib, the leading cause of meningitis and pneumonia, particularly in infants for which non-conjugated www.drugdiscoverytoday.com
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Glossary BSA: bovine serum albumin, a model protein commonly used to test prototype vaccine. CPS: capsular polysaccharide, the serotype common antigen of bacteria. EDC: N-ethyl-N0 -(3-dimethylaminopropyl)carbodiimide, a water-soluble peptide coupling reagent. Epitope: a part of an antigen to which an antibody binds. Also called an antigenic determinant. GM2, GD2, GD3: mono- or di-sialylated gangliosides, tumor-associated, lipidic carbohydrate antigens. GMP: good manufacturing practice. GPI: glycosylphosphatidylinositol, linker in certain cell membrane glycoproteins. Hib: Haemophilus inflenza type b. Immunoadjuvant: a non-specific substance acting to enhance the immune response to an antigen with which it is administered. KLH: keyhole limpet hemocyanin, a high molecular weight polymeric glycoprotein derived from the hemolymph of the giant sea mollusk Megathura crenulata frequently (and erroneously) used as a highly immunogenic carrier for carbohydrate antigens. MAb: monoclonal antibody. MHC: major hystocompatibility complex, a complex of proteins on antigen presenting cells wherein the antigens are bounds and exposed to T-cell receptors. PADRE: pan HLA-DR epitope, a universal mice T-cell epitope used as immunogenic carrier.
vaccines failed, the disease has been essentially eradicated where national vaccination programs have been implemented. This review will highlight recent trends in carbohydratebased vaccines using target antigens following their historical developments. A brief overview of strategies is initially presented followed by their applications in: bacterial CPS, parasite vaccines, HIV gp120-directed vaccines, and cancer vaccines. The closing sections describe approaches toward future vaccines and comparisons of existing technologies with their advantages and disadvantages.
Key technologies and strategies Historically, carbohydrate-based vaccines, like others, were composed of killed or attenuated bacteria. Nowadays, purified antigens are required, but for these T-cell independent antigens, conjugation to T-cell dependent protein carriers, capable of immune cell activation through MHC (see Glossary) class II-restricted CD4+ T cells, is still critical. This is because the carbohydrates alone do not stimulate memory effects and protection only last for one or two years. Activating B-cell memories to produce high affinity IgG antibodies permits protection for some 10–15 years. Isolation, purification and structural as well as physical characterization of bacterial CPS have become more or less routine with modern machineries. However, it remains that these chemical entities are heterogeneous mixtures of polymers varying in molecular weights, end-groups and degrees of branching and substitutions by key functionalities such as acetate, pivalate and phosphate groups that can modulate the level of immunogenicities [1]. 328
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From an analytical and Epitope (see Glossary) mapping point of view, the above heterogeneity factors are even more complex when dealing with glycoproteins, lipopolysaccharides and glycans from parasites (glycosylphosphatidylinositol (GPI) anchors, see Glossary). Additionally, for carbohydrate-based cancer vaccines, usually originating from O-linked glycans, the common immunodominant epitopes, although cryptic in healthy tissues and overlay expressed on cancer cells, are still ubiquitous self-antigens that further complicate the mounting of an effective and selective immune response which might lead to autoimmune disease. With increasing and more stringent regulatory issues for new vaccines approval, it is clear that welldefine synthetic carbohydrate antigens are becoming appealing. A comparison Table 1 illustrates the various approaches described in the design of carbohydrate-based vaccines.
Capsular polysaccharide protein conjugate-based vaccines Bacterial capsular polysaccharide vaccines Despite the deficiencies of bacterial CPS (see Glossary) to mount efficient immune responses in infants, they are still considered the most attractive targets since they have highly conserved structures shown to provide a reliable source of bactericidal antibodies. This is why, their conjugation to protein carriers have lead to successful vaccines and candidate vaccines [1–5]. The success encountered with several vaccines composed of the natural polysaccharide Hib, conjugated to different protein carriers (Vaxem-Hib1 (Chiron, http://www.chiron.com/), Hiberix1 (Glaxo Smith Kline Biologicals, http://www.gsk.com/index.htm)) was therefore and ideal situation to bring the technologies to higher level of sophistications. A team of Cuban and Canadian scientists has recently succeeded in preparing a fully synthetic polysaccharide epitope on a large scale and under GMP conditions (see Glossary) which after covalent attachment to tetanus toxoid provided a commercial vaccine (Quimi Hib1, Heber Biotec, Cuba) that is equally effective to the existing ones [6]. The synthetic strategy selected in the present case differs from the previous attempts using solid phase stepwise synthesis of the phosphodiester linkages [7–9]. Rather, oligomerization of synthetic fragments comprising end groups functionalized as H-phosphonate derivatives was achieved in a single step reaction to provide, under controlled conditions, a synthetic polysaccharide possessing between six and nine average repeating units. The unique linking arm was then covalently bound to a thiolated protein using the conjugate addition to a maleimido function. Thus, once in hand, the repeating units are condensed in a single step process rather than the 16 or so steps required by solid-phase chemistry. The structure and the linking arm of this first semi-synthetic vaccine are illustrated in Fig. 1a.
Kihlberg group in Sweden
Heterogeneous in the carbohydrate complex
Epimmune Inc
[10,11]
Heterogeneous in the protein complex
Heber Biotec (Cuba); Biomira; Sloan-Kettering Inst. for Cancer Research; Anchora Pharmaceuticals; Optimer Pharmaceuticals
[5,6,15,16,25] [2–4] [1] References
Aventis Pasteur Chiron GlaxoSK Merck & Co Aventis Pasteur Groups and companies using it
Complex analysis/ purification Immunity only last 2–5 years, potential contamination by bacterial components Cons
[46–48]
Unknown
Specific immune response Large scale access, ease of quality control Immunity last 10–15 years Easy access Pros
Research only PADRE1 Quimi-Hib1 Theratope1 MenactraTM Vaxem-Hib1 Hiberix1 Pneumovax1 Menomune1 Specific product names
Fully synthetic vaccines Bacterial PS- synthetic T-cell epitopes Synthetic antigen–protein conjugate Bacterial PS–protein conjugate Bacterial polysaccharide (PS) Approaches
Table 1. Comparison summary table
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Purer material, quality control easy
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The technology may soon reach the level of fully synthetic vaccines wherein even the protein epitope could be of synthetic origin. Indeed, candidate vaccines composed of natural CPS from S. pneumoniae serotypes 14, 6B and 9V were prepared by attaching these chemically modified polysaccharides to a universal 13 amino acid helper T-lymphocyte epitope (Pan HLA-DR Epitope (PADRE, see Glossary)) [10]. This peptide, AKXVAAWTLKAAA (X = cyclohexylalanine), binds to murine I-Ab molecules in the T-cell receptor and is immunogenic in C57BL/6 mice. Activation of the polysaccharides (PS, MWave 15,000, the average molecular weight) with cyanogen bromide and treatment with adipic acid dihydrazide provided precursors linked to the synthetic peptide by carbodiimide (EDC) coupling. When adsorbed onto Al(OH)3, the PADRE–PS6B conjugate vaccine produced IgG1 isotypes that were bactericidal. Unfortunately, the cyanogen bromide activation of the PS is a random process with multiple point attachment, therefore diminishing the criteria of well-define chemicals. Fig. 1b illustrates a representative structure of the above semi-synthetic PADRE–PS6B conjugate. A universal peptide epitope derived from tetanus toxin (TT230–244) was also previously shown to be useful in 95% of the human population, thus illustrating further the potential for entirely synthetic vaccine candidates using a combination of the above two strategies [11–14].
Synthetic carbohydrate antigens protein conjugates Parasite vaccines Two other major breakthroughs in synthetic carbohydrate chemistry are responsible for the potential achievements of other semi-synthetic vaccines. In one case, the programmable, one-pot, solution-phase synthesis technique (Optimer Pharmaceuticals, San Diego: http://www.optimerpharma.com/) makes it possible to rapidly and efficiently prepare sizable amounts of complex oligosaccharides [15]. In the second case, Anchora Pharmaceuticals (Cambridge, MA) is exploiting an automated solid-phase synthetic methodology developed by Seeberger (http://www.ethlife.ethz.ch/cd/bits/ whoswho/prof/engl/1683engl.html) and co-workers to provide access to carbohydrate epitopes that can be incorporated into potential vaccines against malaria, HIV, tuberculosis and bacterial infections [16]. The above technologies are being put in practice against protozoans (Plasmodium falciparum (malaria), Trypanosoma cruzi (Chagas disease), Leishmania donovani) and helminthic parasites (worms) such as Schistosoma mansoni [17,18]. For instance, Schofield and Seeberger recently synthesized P. falciparum derived GPI which, when coupled to immunogenic protein carriers (Fig. 2a) afforded a vaccine that protected mice against malaria acidosis, pulmonary edema, cerebral syndrome and fatality [19]. Although none destructive for the bacteria itself, the vaccine seems to have all the properties of anti-toxic activities. GPIs from T. cruzi and T. www.drugdiscoverytoday.com
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Figure 1. (a) Structure of the first approved semi-synthetic commercial carbohydrate-based vaccine containing the Haemophilus influenzae type b capsular polysaccharide linked to the immunogenic protein carrier Tetanus toxoid. Here a fully synthetic scheme provided the entire portion of the carbohydrate epitope as opposed to the more classical bacteria-isolated polysaccharide [6]. (b) Likely structure of the murine vaccine composed of the capsular polysaccharide from Streptococcus pneumoniae serotype 6B conjugated to the universal helper T-lymphocyte epitope (PADRE1) using cyanogen bromide activation of the polysaccharide, reaction with adipic acid dihydrazide, followed by coupling to the peptide with a carbodiimide (EDC) [10].
brucei have analogous toxins, suggesting that they can be proinflammatory agents. Importantly, human GPIs differ sufficiently from those of the parasites that cross-reactivity is unlikely. Combinations of chemo-enzymatic strategies also provide clear advantages over more classical synthetic approaches to gain access into complex glycan structures. Similarly, to the above cases, a conjugate vaccine against schistosomiasis and perhaps other parasitic helminthes was prepared by using schistosome glycan antigens [20]. The strategy involved preparing ‘core glycopeptide’ from commercially available human fibrinogen by pronase digestion, remodeling the 330
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glycan structure by sequential glycosidases, followed by treatment with recombinant glycosyltransferase derived from the worms to generate the required antigenic parasite glycan. Fig. 2b shows an example of such vaccine wherein the schistosomes LacdiNAc (LDN) conjugate antigen on complex-type biantennary N-glycans was chemo-enzymatically prepared. The vaccine was then synthesized by treating the free asparagine amine with iminothiolane (Traut’s reagent) that provided a thiol end-group which subsequently reacted with maleimide-bovine serum albumin (BSA, see Glossary) resulting in an amidine conjugate. Mice immunization afforded IgGs.
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Figure 2. (a) Prototype carbohydrate anti-toxic vaccine against the malaria parasite Plasmodium falciparum linked to KLH or ovalbumin (OVA) [19]. (b) Schistosoma vaccine prepared by chemo-enzymatic glycan remodeling of human glycoprotein and covalent attachment to BSA [20].
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HIV gp120 vaccines The failures to elicit potent and widely protective immune responses against HIV in AIDS patients might soon be overcome with the advent of information resulting from the X-ray structural data of Calarese et al. [21]. The crystal data showed the structures of the human antibody 2G12, one of the most potent neutralizing antibody known so far, bound to a unique carbohydrate antigen on HIV-1 gp120. A conserved N-linked glycopeptide region on gp120 at residues N332, N339 and at N392 was associated with MAb 2G12 at the two primary binding sites and one at an unusual VH/VH’ interface. A modeled structure revealed that the apex of a triangle formed between the above asparagine residues in ˚ , 18.25 A ˚ and 12.2 A ˚ apart, gp120 [22] were found 20.3 A respectively [23,24]. These useful informations have triggered four different research groups to tackle the daunting task of preparing the required complex triantennary oligomannosides [22–29]. Thus, Danishefky et al. [25–27] as chosen their efficient but stepwise glycal assembly method to synthesize a nonasaccharide glycopeptide, Seeberger et al. used their solid-phase protocol [28], Wong et al. employed their one-pot, solution-phase synthesis technique [29], while finally, Li and Wang used a chemoenzymatic approach that readily provided Man9GlcNAc2Asn undecasaccharide from pronase degradation of soybean agglutinin [23,24]. They then attached three of the above Man9GlcNAc2Asn residues to a cholic acid scaffold, thus affording a trimeric mimic of the HIV gp120 glycoprotein. The construct showed an IC50 of 21 mM in comparison to 960 mM for the monovalent Man9GlcNAc2Asn epitope in a competitive solid-phase inhibition assay using gp120 as coating antigen and MAb 2G12, together with horseradish peroxidase-labeled goat anti-human IgG. This result compared relatively well to the inhibition by gp120 itself, albeit a nanomolar inhibitor. Interestingly, the cholic acid scaffold was built with a terminal amine functionality for further attachment to an immunogenic protein carrier.
Toward fully synthetic vaccines Cancer vaccines In contrast to normal cells, the O-linked glycoprotein patterns of mucins (mainly MUC1 and 4), gangliosides (GM2, GD2 and GD3, see Glossary), and neutral glycolipids (globo-H and Lewisy) on epithelial tumor cells are dramatically altered both in number and in structures [30–32]. In some of the socalled tumor-associated carbohydrate antigens of mucins, a down-regulation of a key b-1,6-GlcNAc transferase, causes an accumulation of the much shorter core1 glycan chain b-Gal(1 ! 3)-a-GalNAc-O-Ser/Thr (referred to as the T- or TF-antigen (TF = Thomsen–Friedenreich)). In the presence, in healthy tissues, of the above enzyme, completion of glycan biosynthesis provides a much more complex pattern of glycation, amounting 50% of the glycoprotein masses. The 332
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consequences of these modified glycosylation profiles are profound as they contribute to MUC1 and MUC4 conformational changes, better exposition of peptide backbones, and obviously to an over accumulation of the above carbohydrate-based cancer markers which are otherwise cryptic (masked) on healthy tissues. Furthermore, large amount of the a-GalNAc-O-Ser/Thr (Tn-antigen) precursor is also found accumulated together with the corresponding sialylated counterparts (sialyl T- or sialyl Tn antigens (ST and STn)) because an 8- to 10-fold increase activity of sialyltransferases in tumor cells. Consequently, most efforts to provide carbohydrate-based vaccines have been devoted with limited success to the above antigens. A breast cancer vaccine in clinical Phase III trial (Theratope1, Biomira Inc, Edmonton), composed of synthetic clusters of STn built on tandem repeat of the MUC1 glycopeptide and further attached to a highly immunogenic, but ill-define keyhole limpet hemocyanin glycoprotein (KLH, see Glossary), failed to provide the expected protection [33]. However, it appears to benefit a subpopulation of patients taking hormonal therapy before the trial. It is our opinion that, although widely used in vaccine preparation, KLH should be entirely avoided as an immunogenic protein carrier as it was shown to contain epitopes from schistosoma genus [20]. Furthermore, its high molecular weight (<1000 kDa) preclude detailed composition analysis of the ensuing conjugate vaccines. Another potential source of failure for cancer vaccines may rely on their incomplete immune responses that one can mount against the glycoconjugates. Perhaps a key to the problem is to look for not only an humoral, antibody-based response but also for cytotoxic T-helper assistance that requires MHC-class I CD8+ subsets. In this regard, a recent paper by Dziadek and Kunz [34] describes a fully synthetic preparation by solid-phase synthesis of sialyl-Tn-glycodecapeptide containing both a fragment of the peptide repeat motif of MUC1 (HGVTSAPDTRPAPGSTAPPA) and a T-cell epitope from tetanus toxin linked by a spacer (Fig. 3a). In a T-cell proliferation assay, 53% of the T-cell produced were the desired cytotoxic CD8+. The paper did not discussed tumoricidal activity. Alternatively, it is also possible to prepare a vaccine with several simultaneous B-cell carbohydrate epitopes. This is at least the approach recently taken by Danisefsky and his team at the Memorial Sloan-Kettering Cancer Center [35–39]. In this approach, using their synthetic glycal strategy, they attached five different carbohydrate-associated tumor markers to KLH (Fig. 3b). In this way, the simultaneous generation of several specific antibodies may leave very little chances for cancer cells to survive. In a different clinical Phase II study including only the Globo H antigen–KLH conjugate [40], this group found the vaccine to induce complement-dependent cytotoxicity. The patients were found to
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Figure 3. Semi-synthetic carbohydrate-associated tumor vaccines. (a) Synthetic glycopeptide comprising a repeat motif of the O-linked glycoprotein mucin type 1 bearing the sialyl-Tn carbohydrate antigen (MUC1) and a universal T-cell epitope derived from tetanus toxin [34]. (b) A multivalent prototype cancer vaccine bearing several of the most common carbohydrate-associated tumor markers linked to keyhole lympet hemocyanin (KLH) [35,36].
have IgM and IgG titers superior to what is normally achieved from bacterial CPS vaccines. Using their synthetic approach, affinity chromatography matrix could also be provided, thus allowing isolation and quantitation of antibodies. Importantly, they suggested that the patients’ own purified antibodies would be useful therapeutics after either radiolabeling
or drug-conjugation. Such an approach sounds very appealing from a regulatory point of view since it would avoid dealing with ‘humanized’ mouse monoclonal antibodies. Automated solid-phase syntheses of some of the above carbohydrate antigens has also been achieved, thus further expanding the scope of cancer vaccines [41]. www.drugdiscoverytoday.com
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Links
Figure 4. Idealized fully synthetic carbohydrate-based vaccine containing several carbohydrate and immunogenic T-cell epitopes which would most likely be useful against cancer cells, family of bacteria or parasites. Suitable T-cell epitopes are short, usually 15-mer peptides, recognized by most human Major Histocompatibily Complexes (MHC Class II). To reach most individuals, more than one allele-dependent T-cell epitope is required. An example of almost universal human T-cell epitope is tetanus toxin fragment 830–843 [11–14].
Non-carbohydrate anti-carbohydrate-based vaccines Future vaccines It is not unlikely that future glycan-based vaccines would ironically not contain any carbohydrate structures. The reasons for this is that there is an increasing body of evidences suggesting that carbohydrate mimotopes can be generated out of peptide sequences. The origin for this relatively novel strategy is based on the many observations that anti-idiotypic antibodies, provided peptide sequences that were mimicking the structures of carbohydrates. There are now several cases at hand demonstrating that antibodies generated from carbohydrate peptide-mimics could recognize and bind to bacterial CPS [42]. Two such good examples were provided by Pinto et al. (http://www.sfu.ca/chemistry/faculty/pinto.htm) with Streptococcus Group A and Shigella flexneri [43–45]. These findings are valuable in that readily prepared peptides acting as carbohydrate mimics, once attached to universal T-cell peptide epitopes, should provide entirely synthetic vaccines. Fundamental understandings on how the human immune system handles carbohydrate antigens is of prime importance for the future design of simple, ideally totally synthetic, carbohydrate-based vaccines. One such research activity is being actively pursued by the group of Kihlberg et al. in Sweden ([email protected]) who is evaluating the paramaters that could ultimately lead to rheumatoid arthritis vaccines [46–48].
Conclusions and comparisons of technology The rapid growth of sophisticated analytical tools, purification instruments, and proteo/glyco-mics allows the structural 334
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Sloan-Kettering Institute: carbohydrate-based antitumor vaccines: http://www.mskcc.org/mskcc/html/11263.cfm The development of anti-cancer carbohydrate vaccines: http:// www.nd.edu/aostafin/benl/2000/page5.html http://pubs.acs.org/isubscribe/journals/cen/78/i35/html/ 7835notw8.html Neisseria Meningitidis, mode of infection: http://www.brown.edu/ Courses/Bio_160/Projects1999/bmenin/nmenin.html Optimer Programmed One-Pot Synthesis Technology-OPopS(TM) Technology: http://www.optimerpharma.com/core_technology.htm Phase III Theratope1 vaccine trial: http://www.biomira.com/news/ detailNewsRelease/166/%3E PADRE carrier/adjuvant: http://www.epimmune.com/technology/ padre.cfm Aventis MenactraTM vaccine for protection against meningococcal infection. First candidate quadrivalent conjugate meningococcal vaccine: http://www.aventispasteur.com/index.cfm?FA=25022004
indentifications of an increasing number of target carbohydrate antigens of microbial origins and disease states. When coupled to our rising arsenals of chemo-enzymatic skills to build complex carbohydrate structures, including CPS, and our understanding of immune processing to such carbohydrate antigens, it is likely that we will witness more and better carbohydrate-based vaccines. It makes no doubt that critical milestones reside in recent progress achieved in synthetic carbohydrate and organic chemistry. Modern, stepwise and multistep syntheses of complex carbohydrates, although efficient in providing clean compounds, are usually to cumbersome for practical and largescale applications. Hence, the progress made by the one-pot solution-phase or solid-phase syntheses are likely to surpass more classical approaches. The above strategies, when coupled to well-established solid-phase peptide syntheses of immunogenic T-cell epitopes, are amenable to scale-up processes that will generate large amount of the small molecular weight candidate vaccines. As discussed above, fully synthetic vaccines are easier to characterized, handle, and are thus more likely to be rapidly approved by health organisms such as the Food and Drug Administration (FDA).
Related articles Danishefsky, S.J. and Allen, J.R. (2000) From the laboratory to the clinic: a retrospective on fully synthetic carbohydrate-based anticancer vaccines. Angew. Chem. Int. Ed. 39. 836–863 Borman, S. (2004) Carbohydrate vaccines. C&EN August 9, 31–34 (http:// pubs.acs.org/cen/coverstory/8232/8232vaccines.html) Borman, S. (2002) Vaccines by automated synthesis. C&EN January 21, 43–44 (http://pubs.acs.org/isubscribe/journals/cen/80/i03/html/ 8003sci4.html) Keding, S.J. and Danishefsky, S.J. (2003) Synthetic carbohydrate-based vaccines. Carbohydr. Based Drug Discov. 1, 381–406
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Not withstanding the recent success encountered with the semi-synthetic Hib vaccine, the complexity of bacterial polysaccharides are not always amenable to similar one-pot oligomerization. In the above case, the simple repeating units were linked together by a one-step, solution-phase phosphodiester assembly, similarly to those observed in oligonucleotides but not widely distributed. The situation could be even more complicated when several complex repeating units, se pentasaccharides, are required to form a conformational epitope that is reached when only half a dozen such structures are assembled. It is our belief that the ideal vaccines of the future will be constituted of synthetic carbohydrate/peptide antigens assembled on an equally synthetic small immunogenic peptides. Because of the complexity of the human T-cells, it is also likely that several T-cell peptide epitopes would be required to mount a universal vaccine. As vaccination for infants in developing countries is not a simple task, multivalent vaccines or cocktail of vaccines is highly recommended. The structure of an idealized vaccine is illustrated in Fig. 4. Equally important could be the combination of various antigens into liposomes for which the immunoadjuvant properties have long been recognized [49,50].
Outstanding issues Are productions of synthetic bacterial capsular polysaccharides cost effective compared to isolation from bacterial cultures? Are governmental health regulations more likely to accept synthetic vaccines in comparison to those obtained from bacteria? Are their truly universal T-cell epitopes? Totally synthetic vaccines, myth or reality?
References 1 Dick, W.E. Jr and Beurret, M. (1989) Glycoconjugates of bacterial carbohydrate antigens. In Contribution to Microbiology and Immunnology: Conjugate Vaccines (Cruse, J.M. and Lewis, Jr, R.E. (eds.), pp. 48–114, S. Karger 2 Jennings, H.H. and Pon, R.A. (1996) Polysaccharides and glycoconjugates as human vaccines. In Polysaccharides in medicinal applications (Dumitriu, S. ed.), pp. 443–479, Marcel Dekker 3 Robbins, J.B. et al. (1999) Bacterial polysaccharide-protein conjugate vaccines. Pure Appl. Chem. 71, 745–754 4 Moreau, M. and Schulz, D. (2000) Polysaccharide Based Vaccines for the prevention of pneumococcal infections. J. Carbohydr. Chem. 19, 419–434 5 Pozsgay, V. (2000) Oligosaccharide-protein conjugates as vaccine candidates against bacteria. Adv. Carbohydr. Chem. Biochem. 56, 153–200 6 Verez-Bencomo, V. et al. (2004) A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science 305, 522–525 7 Peeters, C.C. et al. (1992) Synthetic trimer and tetramer of 3-beta-Dribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys. Infect. Immun. 60, 1826–1833 8 Nilsson, S. et al. (1992) Solid-phase synthesis of a fragment of the capsular polysaccharide of Haemophilus influenzae type B using H-phosphonate intermediates. J. Carbohydr. Chem. 11, 265–285 9 Kandil, A.A. et al. (1992) Synthesis of fragments of the capsular polysaccharide of Haemophilus influenzae type b on soluble polymeric support. Synlett 7, 555–557
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10 Alexander, J. et al. (2004) Development of experimental carbohydrateconjugate vaccines composed of Steptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE1). Vaccine 22, 2362–2367 11 Falugi, F. et al. (2001) Rationally designed strings of promiscuous CD4+ Tcell epitopes provides help to Haemophilus influenza type b oligosaccharide: a model for new conjugate vaccines. Eur. J. Immunol. 31, 3816–3824 12 Diethelm-Okita, B.M. et al. (1997) Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments. J. Infect. Dis. 175, 382–391 13 Demotz, S. et al. (1993) The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86. Eur. J. Immunol. 23, 425–432 14 Valmori, D. et al. (1992) Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. J. Immunol. 149, 717–721 15 Koeller, K.M. and Wong, C.H. (2000) Synthesis of complex carbohydrates and glycoconjugates: enzyme-based and programmable one-pot strategies. Chem. Rev. 100, 4465–4493 16 Plante, O.J. and Seeberger, P.H. (2003) Recent advances in automated solid-phase carbohydrate synthesis: from screening to vaccines. Curr. Opin. Drug Discov. Dev. 6, 521–525 17 Seeberger, P.H. et al. (2004) A convergent, versatile route to two synthetic conjugate anti-toxin malaria vaccines. Chem. Commun. 15, 1706–1707 18 Hewitt, M.C. et al. (2002) Rapid synthesis of a glycosylphosphatidylinositol-based malaria vaccine using automated solid-phase oligosaccharide synthesis. J. Am. Chem. Soc. 124, 13434–13436 19 Schofield, L. et al. (2002) Synthetic GPI as candidate anti-toxic vaccine in a model of malaria. Nature 418, 785–789 20 Nyame, A.K. et al. (2004) Antigenic glycans in parasitic infections: implications for vaccines and diagnostics. Arch. Biochem. Biophys. 426, 182–200 21 Calarese, D.A. et al. (2003) Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071 22 Wyatt, R. et al. (1998) The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393, 705–711 23 Li, H. and Wang, L-X. (2004) Design and synthesis of a templateassembled oligo-mannose cluster as an epitope mimic for human HIVneutralizing antibody 2G12. Org. Biomol. Chem. 2, 483–488 24 Wang, L-X. et al. (2004) Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12. Implications for HIV-1 vaccine design. Chem. Biol. 11, 127–134 25 Dudkin, V.Y. et al. (2004) Toward fully synthetic carbohydrate-based HIV antigen design: on the critical role of bivalency. J. Am. Chem. Soc. 126, 9560–9562 26 Mandal, M. et al. (2004) Glycopeptides (1): in pursuit of carbohydratebased HIV vaccines, part 1: the total synthesis of hybrid-type gp120 fragments. Angew. Chem. Int. Ed. 43, 2557–2561 27 Geng, X. et al. (2004) Glycopeptides (2): in pursuit of carbohydrate-based HIV vaccines, Part 2: the total synthesis of high-mannose-type gp120 fragments-evaluation of strategies directed to maximal convergence. Angew. Chem. Int. Ed. 43, 2562–2565 28 Adams, E.W. et al. (2004) Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology glycan-dependent gp120/protein interactions. Chem. Biol. 11, 875–881 29 Lee, H-K. et al. (2004) Reactivity-based one-pot synthesis of oligomannoses: Defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody. Angew. Chem. Int. Ed. 43, 1000–1003 30 Ragupathi, G. (1996) Carbohydrate antigens as targets for active specific immunotherapy. Cancer Immunol. Immunother. 43, 152–157 31 Livingston, P.O. et al. (1997) Carbohydrate vaccines that induce antibodies against cancer: rationale. Cancer Immunol. Immunother. 45, 1–9 32 Lloyd, K.O. (2000) Carbohydrate vaccines for the immunotherapy of cancer. Drug News Perpect. 13, 463–470 33 Reddish, M.A. et al. (1997) Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes. Glycoconj. J. 14, 549–560 34 Dziadek, S. and Kunz, H. (2004) Synthesis of tumor-associated glycopeptide antigens for the deveolpment of tumor-selective vaccines. Chem. Record. 3, 308–321
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35 Ragupathi, G. et al. (2003) A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers. Cancer Immunol. Immunother. 52, 608–616 36 Ragupathi, G. et al. (2002) On the power of chemical synthesis: immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines. Proc. Natl. Acad. Sci. USA 99, 13699–13704 37 Allen, J.R. and Danishefsky, S.J. (2001) Carbohydrate-based tumor antigens as antitumor vaccine agents. ACS Symp. Ser. 796, 299–316 38 Kudryashov, V. et al. (2001) Toward optimized carbohydrate-based anticancer vaccines: epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice. Proc. Natl. Acad. Sci. USA 98, 3264–3269 39 Allen, J.R. et al. (2001) Pursuit of optimal carbohydrate-based anticancer vaccines: preparation of a multiantigenic unimolecular glycopeptide containing the Tn, MBr1, and Lewisy antigens. J. Am. Chem. Soc. 123, 1890– 1897 40 Wang, et al. (2000) Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: Isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens. Proc. Natl. Acad. Sci. USA 97, 2719–2724 41 Routenberg Love, K. and Seeberger, P.H. (2004) Automated solid-phase synthesis of protected tumor-associated antigen and blood-group determinant oligosaccharides. Angew. Chem. Int. Ed. 43, 602–605
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42 Johnson, M.A. and Pinto, B.M. (2002) Molecular mimicry of carbohydrates by peptides. Aust. J. Chem. 55, 13–25 43 Vyas, N.K. et al. (2003) Structural basis of peptide-carbohydrate mimicry in an antibody-combining site. Proc. Natl. Acad. Sci. USA 100, 15023– 15028 44 Hossany, R.B. et al. (2004) Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines. Bioorg. Med. Chem. 12, 3743–3754 45 Johnson, M.A. et al. (2003) Rational design and synthesis of peptide ligands for an anti-carbohydrate antibody and their immunochemical characterization. Bioorg. Med. Chem. 11, 781–788 46 Holm, B. et al. (2003) Role of the galactosyl moiety of collagen glycopeptides for T-cell stimulation in a model for rheumatoid arthritis. Bioorg. Med. Chem. 11, 3981–3987 47 Mogemark, M. et al. (2003) Influence of saccharide size on the cellular immune response to glycopeptides. Org. Biomol. Chem. 1, 2063–2069 48 Holm, B. et al. (2002) Glycopeptide specificity of helper T cells obtained in mouse models for rheumatoid arthritis. Chem. Biol. Chem. 3, 1209–1222 49 Samuel, J. et al. (1998) Immunogenicity and antitumor activity of a liposomal MUC1 peptide-based vaccine. Int. J. Cancer 75, 295–302 50 Guan, H.H. et al. (1998) Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses. Bioconj. Chem. 9, 451–458
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Lead optimization
New trends in carbohydrate-based vaccines Rene´ Roy Department of Chemistry, Universite´ du Que´bec a` Montre´al, P.O. Box 8888, Succ. Centre-Ville, Montre´al, Que´bec, Canada H3C 3P8
Carbohydrate-based vaccines constitute successful therapeutic arsenals against microbial infections, particularly since the advent of bacterial capsular polysaccharide (CPS) protein conjugates. These prophylactic approaches are increasingly more accessible for other therapies such as viral, parasitic, fungal infections and even against certain forms of cancer. Three major discoveries are responsible for rapid growth in this field. In the first instance, the recent commercialization of the first semi-synthetic vaccine against bacterial infections caused by Haemophilus influenza type b, second by the rapid access to complex saccharide structures through one-pot, solution-phase syntheses, and third by automated solid-phase synthesis.
Introduction Bacterial, parasitic and viral infections constitute one of the major health problems worldwide. Drug resistance is rapidly escalating and new drug developments, although faster than in the past with the advent of combinatorial methods, is becoming prohibitive. Therefore, the development of new prophylactic and potent therapeutic vaccines to combat these types of infections is of utmost importance, especially in developing countries. The cell-surface of most pathogens and cancer cells express several structurally variable and sometime cryptic carbohydrate molecules that are thus E-mail address: [email protected]. 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.10.005
Section Editor: Constant A.A. van Boeckel – Organon NV, Oss, The Netherlands The capsular polysaccharides of various pathogenic bacteria have been used for many decades as vaccines. Later it turned out that such vaccines are more efficacious, in particular in childhood, if they are applied as conjugates with non-endogenous proteins. During the past decade, several synthetic methodologies have been developed to prepare well-defined carbohydrate epitopes, which are much smaller than the native polysaccharides and which can be hooked via synthetic linkers to the desired protein. Moreover, peptide synthesis allows the preparation of tailor-made peptide fragments comprising so-called Tcell epitopes. By using various carbohydrate, peptide and conjugation technologies, fully synthetic vaccines can be developed. Such technologies open new avenues not only for bacterial and viral vaccines but also for novel anti-cancer vaccines. Rene´ Roy has long-standing experience in carbohydrate chemistry and conjugation of complex synthetic epitopes to produce synthetic clusters and vaccines. He is one of the contributors towards the first semi-synthetic vaccine against Haemophilus influenza type b that is efficacious in human beings.
exposed at the forefront battle against the immune system. Hence, by triggering a specific and a high-affinity immune response against a particular cell surface carbohydrate antigen, protective and long lasting immunity is thus achieved. This strategy has been very successful with bacterial capsular polysaccharides (CPS) and several such glycoconjugates are now commercially available against Neisseria meningitidis groups A, C, W-135 and Y, Steptococcus pneumoniae (23 serotypes), Haemophilus influenza type b (Hib, see Glossary) and Salmonella typhi [1–5]. For instance, with the advent in the 1990s of protein conjugate vaccines against the Gram-negative organisms Hib, the leading cause of meningitis and pneumonia, particularly in infants for which non-conjugated www.drugdiscoverytoday.com
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Glossary BSA: bovine serum albumin, a model protein commonly used to test prototype vaccine. CPS: capsular polysaccharide, the serotype common antigen of bacteria. EDC: N-ethyl-N0 -(3-dimethylaminopropyl)carbodiimide, a water-soluble peptide coupling reagent. Epitope: a part of an antigen to which an antibody binds. Also called an antigenic determinant. GM2, GD2, GD3: mono- or di-sialylated gangliosides, tumor-associated, lipidic carbohydrate antigens. GMP: good manufacturing practice. GPI: glycosylphosphatidylinositol, linker in certain cell membrane glycoproteins. Hib: Haemophilus inflenza type b. Immunoadjuvant: a non-specific substance acting to enhance the immune response to an antigen with which it is administered. KLH: keyhole limpet hemocyanin, a high molecular weight polymeric glycoprotein derived from the hemolymph of the giant sea mollusk Megathura crenulata frequently (and erroneously) used as a highly immunogenic carrier for carbohydrate antigens. MAb: monoclonal antibody. MHC: major hystocompatibility complex, a complex of proteins on antigen presenting cells wherein the antigens are bounds and exposed to T-cell receptors. PADRE: pan HLA-DR epitope, a universal mice T-cell epitope used as immunogenic carrier.
vaccines failed, the disease has been essentially eradicated where national vaccination programs have been implemented. This review will highlight recent trends in carbohydratebased vaccines using target antigens following their historical developments. A brief overview of strategies is initially presented followed by their applications in: bacterial CPS, parasite vaccines, HIV gp120-directed vaccines, and cancer vaccines. The closing sections describe approaches toward future vaccines and comparisons of existing technologies with their advantages and disadvantages.
Key technologies and strategies Historically, carbohydrate-based vaccines, like others, were composed of killed or attenuated bacteria. Nowadays, purified antigens are required, but for these T-cell independent antigens, conjugation to T-cell dependent protein carriers, capable of immune cell activation through MHC (see Glossary) class II-restricted CD4+ T cells, is still critical. This is because the carbohydrates alone do not stimulate memory effects and protection only last for one or two years. Activating B-cell memories to produce high affinity IgG antibodies permits protection for some 10–15 years. Isolation, purification and structural as well as physical characterization of bacterial CPS have become more or less routine with modern machineries. However, it remains that these chemical entities are heterogeneous mixtures of polymers varying in molecular weights, end-groups and degrees of branching and substitutions by key functionalities such as acetate, pivalate and phosphate groups that can modulate the level of immunogenicities [1]. 328
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From an analytical and Epitope (see Glossary) mapping point of view, the above heterogeneity factors are even more complex when dealing with glycoproteins, lipopolysaccharides and glycans from parasites (glycosylphosphatidylinositol (GPI) anchors, see Glossary). Additionally, for carbohydrate-based cancer vaccines, usually originating from O-linked glycans, the common immunodominant epitopes, although cryptic in healthy tissues and overlay expressed on cancer cells, are still ubiquitous self-antigens that further complicate the mounting of an effective and selective immune response which might lead to autoimmune disease. With increasing and more stringent regulatory issues for new vaccines approval, it is clear that welldefine synthetic carbohydrate antigens are becoming appealing. A comparison Table 1 illustrates the various approaches described in the design of carbohydrate-based vaccines.
Capsular polysaccharide protein conjugate-based vaccines Bacterial capsular polysaccharide vaccines Despite the deficiencies of bacterial CPS (see Glossary) to mount efficient immune responses in infants, they are still considered the most attractive targets since they have highly conserved structures shown to provide a reliable source of bactericidal antibodies. This is why, their conjugation to protein carriers have lead to successful vaccines and candidate vaccines [1–5]. The success encountered with several vaccines composed of the natural polysaccharide Hib, conjugated to different protein carriers (Vaxem-Hib1 (Chiron, http://www.chiron.com/), Hiberix1 (Glaxo Smith Kline Biologicals, http://www.gsk.com/index.htm)) was therefore and ideal situation to bring the technologies to higher level of sophistications. A team of Cuban and Canadian scientists has recently succeeded in preparing a fully synthetic polysaccharide epitope on a large scale and under GMP conditions (see Glossary) which after covalent attachment to tetanus toxoid provided a commercial vaccine (Quimi Hib1, Heber Biotec, Cuba) that is equally effective to the existing ones [6]. The synthetic strategy selected in the present case differs from the previous attempts using solid phase stepwise synthesis of the phosphodiester linkages [7–9]. Rather, oligomerization of synthetic fragments comprising end groups functionalized as H-phosphonate derivatives was achieved in a single step reaction to provide, under controlled conditions, a synthetic polysaccharide possessing between six and nine average repeating units. The unique linking arm was then covalently bound to a thiolated protein using the conjugate addition to a maleimido function. Thus, once in hand, the repeating units are condensed in a single step process rather than the 16 or so steps required by solid-phase chemistry. The structure and the linking arm of this first semi-synthetic vaccine are illustrated in Fig. 1a.
Kihlberg group in Sweden
Heterogeneous in the carbohydrate complex
Epimmune Inc
[10,11]
Heterogeneous in the protein complex
Heber Biotec (Cuba); Biomira; Sloan-Kettering Inst. for Cancer Research; Anchora Pharmaceuticals; Optimer Pharmaceuticals
[5,6,15,16,25] [2–4] [1] References
Aventis Pasteur Chiron GlaxoSK Merck & Co Aventis Pasteur Groups and companies using it
Complex analysis/ purification Immunity only last 2–5 years, potential contamination by bacterial components Cons
[46–48]
Unknown
Specific immune response Large scale access, ease of quality control Immunity last 10–15 years Easy access Pros
Research only PADRE1 Quimi-Hib1 Theratope1 MenactraTM Vaxem-Hib1 Hiberix1 Pneumovax1 Menomune1 Specific product names
Fully synthetic vaccines Bacterial PS- synthetic T-cell epitopes Synthetic antigen–protein conjugate Bacterial PS–protein conjugate Bacterial polysaccharide (PS) Approaches
Table 1. Comparison summary table
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The technology may soon reach the level of fully synthetic vaccines wherein even the protein epitope could be of synthetic origin. Indeed, candidate vaccines composed of natural CPS from S. pneumoniae serotypes 14, 6B and 9V were prepared by attaching these chemically modified polysaccharides to a universal 13 amino acid helper T-lymphocyte epitope (Pan HLA-DR Epitope (PADRE, see Glossary)) [10]. This peptide, AKXVAAWTLKAAA (X = cyclohexylalanine), binds to murine I-Ab molecules in the T-cell receptor and is immunogenic in C57BL/6 mice. Activation of the polysaccharides (PS, MWave 15,000, the average molecular weight) with cyanogen bromide and treatment with adipic acid dihydrazide provided precursors linked to the synthetic peptide by carbodiimide (EDC) coupling. When adsorbed onto Al(OH)3, the PADRE–PS6B conjugate vaccine produced IgG1 isotypes that were bactericidal. Unfortunately, the cyanogen bromide activation of the PS is a random process with multiple point attachment, therefore diminishing the criteria of well-define chemicals. Fig. 1b illustrates a representative structure of the above semi-synthetic PADRE–PS6B conjugate. A universal peptide epitope derived from tetanus toxin (TT230–244) was also previously shown to be useful in 95% of the human population, thus illustrating further the potential for entirely synthetic vaccine candidates using a combination of the above two strategies [11–14].
Synthetic carbohydrate antigens protein conjugates Parasite vaccines Two other major breakthroughs in synthetic carbohydrate chemistry are responsible for the potential achievements of other semi-synthetic vaccines. In one case, the programmable, one-pot, solution-phase synthesis technique (Optimer Pharmaceuticals, San Diego: http://www.optimerpharma.com/) makes it possible to rapidly and efficiently prepare sizable amounts of complex oligosaccharides [15]. In the second case, Anchora Pharmaceuticals (Cambridge, MA) is exploiting an automated solid-phase synthetic methodology developed by Seeberger (http://www.ethlife.ethz.ch/cd/bits/ whoswho/prof/engl/1683engl.html) and co-workers to provide access to carbohydrate epitopes that can be incorporated into potential vaccines against malaria, HIV, tuberculosis and bacterial infections [16]. The above technologies are being put in practice against protozoans (Plasmodium falciparum (malaria), Trypanosoma cruzi (Chagas disease), Leishmania donovani) and helminthic parasites (worms) such as Schistosoma mansoni [17,18]. For instance, Schofield and Seeberger recently synthesized P. falciparum derived GPI which, when coupled to immunogenic protein carriers (Fig. 2a) afforded a vaccine that protected mice against malaria acidosis, pulmonary edema, cerebral syndrome and fatality [19]. Although none destructive for the bacteria itself, the vaccine seems to have all the properties of anti-toxic activities. GPIs from T. cruzi and T. www.drugdiscoverytoday.com
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Figure 1. (a) Structure of the first approved semi-synthetic commercial carbohydrate-based vaccine containing the Haemophilus influenzae type b capsular polysaccharide linked to the immunogenic protein carrier Tetanus toxoid. Here a fully synthetic scheme provided the entire portion of the carbohydrate epitope as opposed to the more classical bacteria-isolated polysaccharide [6]. (b) Likely structure of the murine vaccine composed of the capsular polysaccharide from Streptococcus pneumoniae serotype 6B conjugated to the universal helper T-lymphocyte epitope (PADRE1) using cyanogen bromide activation of the polysaccharide, reaction with adipic acid dihydrazide, followed by coupling to the peptide with a carbodiimide (EDC) [10].
brucei have analogous toxins, suggesting that they can be proinflammatory agents. Importantly, human GPIs differ sufficiently from those of the parasites that cross-reactivity is unlikely. Combinations of chemo-enzymatic strategies also provide clear advantages over more classical synthetic approaches to gain access into complex glycan structures. Similarly, to the above cases, a conjugate vaccine against schistosomiasis and perhaps other parasitic helminthes was prepared by using schistosome glycan antigens [20]. The strategy involved preparing ‘core glycopeptide’ from commercially available human fibrinogen by pronase digestion, remodeling the 330
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glycan structure by sequential glycosidases, followed by treatment with recombinant glycosyltransferase derived from the worms to generate the required antigenic parasite glycan. Fig. 2b shows an example of such vaccine wherein the schistosomes LacdiNAc (LDN) conjugate antigen on complex-type biantennary N-glycans was chemo-enzymatically prepared. The vaccine was then synthesized by treating the free asparagine amine with iminothiolane (Traut’s reagent) that provided a thiol end-group which subsequently reacted with maleimide-bovine serum albumin (BSA, see Glossary) resulting in an amidine conjugate. Mice immunization afforded IgGs.
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Figure 2. (a) Prototype carbohydrate anti-toxic vaccine against the malaria parasite Plasmodium falciparum linked to KLH or ovalbumin (OVA) [19]. (b) Schistosoma vaccine prepared by chemo-enzymatic glycan remodeling of human glycoprotein and covalent attachment to BSA [20].
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HIV gp120 vaccines The failures to elicit potent and widely protective immune responses against HIV in AIDS patients might soon be overcome with the advent of information resulting from the X-ray structural data of Calarese et al. [21]. The crystal data showed the structures of the human antibody 2G12, one of the most potent neutralizing antibody known so far, bound to a unique carbohydrate antigen on HIV-1 gp120. A conserved N-linked glycopeptide region on gp120 at residues N332, N339 and at N392 was associated with MAb 2G12 at the two primary binding sites and one at an unusual VH/VH’ interface. A modeled structure revealed that the apex of a triangle formed between the above asparagine residues in ˚ , 18.25 A ˚ and 12.2 A ˚ apart, gp120 [22] were found 20.3 A respectively [23,24]. These useful informations have triggered four different research groups to tackle the daunting task of preparing the required complex triantennary oligomannosides [22–29]. Thus, Danishefky et al. [25–27] as chosen their efficient but stepwise glycal assembly method to synthesize a nonasaccharide glycopeptide, Seeberger et al. used their solid-phase protocol [28], Wong et al. employed their one-pot, solution-phase synthesis technique [29], while finally, Li and Wang used a chemoenzymatic approach that readily provided Man9GlcNAc2Asn undecasaccharide from pronase degradation of soybean agglutinin [23,24]. They then attached three of the above Man9GlcNAc2Asn residues to a cholic acid scaffold, thus affording a trimeric mimic of the HIV gp120 glycoprotein. The construct showed an IC50 of 21 mM in comparison to 960 mM for the monovalent Man9GlcNAc2Asn epitope in a competitive solid-phase inhibition assay using gp120 as coating antigen and MAb 2G12, together with horseradish peroxidase-labeled goat anti-human IgG. This result compared relatively well to the inhibition by gp120 itself, albeit a nanomolar inhibitor. Interestingly, the cholic acid scaffold was built with a terminal amine functionality for further attachment to an immunogenic protein carrier.
Toward fully synthetic vaccines Cancer vaccines In contrast to normal cells, the O-linked glycoprotein patterns of mucins (mainly MUC1 and 4), gangliosides (GM2, GD2 and GD3, see Glossary), and neutral glycolipids (globo-H and Lewisy) on epithelial tumor cells are dramatically altered both in number and in structures [30–32]. In some of the socalled tumor-associated carbohydrate antigens of mucins, a down-regulation of a key b-1,6-GlcNAc transferase, causes an accumulation of the much shorter core1 glycan chain b-Gal(1 ! 3)-a-GalNAc-O-Ser/Thr (referred to as the T- or TF-antigen (TF = Thomsen–Friedenreich)). In the presence, in healthy tissues, of the above enzyme, completion of glycan biosynthesis provides a much more complex pattern of glycation, amounting 50% of the glycoprotein masses. The 332
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consequences of these modified glycosylation profiles are profound as they contribute to MUC1 and MUC4 conformational changes, better exposition of peptide backbones, and obviously to an over accumulation of the above carbohydrate-based cancer markers which are otherwise cryptic (masked) on healthy tissues. Furthermore, large amount of the a-GalNAc-O-Ser/Thr (Tn-antigen) precursor is also found accumulated together with the corresponding sialylated counterparts (sialyl T- or sialyl Tn antigens (ST and STn)) because an 8- to 10-fold increase activity of sialyltransferases in tumor cells. Consequently, most efforts to provide carbohydrate-based vaccines have been devoted with limited success to the above antigens. A breast cancer vaccine in clinical Phase III trial (Theratope1, Biomira Inc, Edmonton), composed of synthetic clusters of STn built on tandem repeat of the MUC1 glycopeptide and further attached to a highly immunogenic, but ill-define keyhole limpet hemocyanin glycoprotein (KLH, see Glossary), failed to provide the expected protection [33]. However, it appears to benefit a subpopulation of patients taking hormonal therapy before the trial. It is our opinion that, although widely used in vaccine preparation, KLH should be entirely avoided as an immunogenic protein carrier as it was shown to contain epitopes from schistosoma genus [20]. Furthermore, its high molecular weight (<1000 kDa) preclude detailed composition analysis of the ensuing conjugate vaccines. Another potential source of failure for cancer vaccines may rely on their incomplete immune responses that one can mount against the glycoconjugates. Perhaps a key to the problem is to look for not only an humoral, antibody-based response but also for cytotoxic T-helper assistance that requires MHC-class I CD8+ subsets. In this regard, a recent paper by Dziadek and Kunz [34] describes a fully synthetic preparation by solid-phase synthesis of sialyl-Tn-glycodecapeptide containing both a fragment of the peptide repeat motif of MUC1 (HGVTSAPDTRPAPGSTAPPA) and a T-cell epitope from tetanus toxin linked by a spacer (Fig. 3a). In a T-cell proliferation assay, 53% of the T-cell produced were the desired cytotoxic CD8+. The paper did not discussed tumoricidal activity. Alternatively, it is also possible to prepare a vaccine with several simultaneous B-cell carbohydrate epitopes. This is at least the approach recently taken by Danisefsky and his team at the Memorial Sloan-Kettering Cancer Center [35–39]. In this approach, using their synthetic glycal strategy, they attached five different carbohydrate-associated tumor markers to KLH (Fig. 3b). In this way, the simultaneous generation of several specific antibodies may leave very little chances for cancer cells to survive. In a different clinical Phase II study including only the Globo H antigen–KLH conjugate [40], this group found the vaccine to induce complement-dependent cytotoxicity. The patients were found to
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Figure 3. Semi-synthetic carbohydrate-associated tumor vaccines. (a) Synthetic glycopeptide comprising a repeat motif of the O-linked glycoprotein mucin type 1 bearing the sialyl-Tn carbohydrate antigen (MUC1) and a universal T-cell epitope derived from tetanus toxin [34]. (b) A multivalent prototype cancer vaccine bearing several of the most common carbohydrate-associated tumor markers linked to keyhole lympet hemocyanin (KLH) [35,36].
have IgM and IgG titers superior to what is normally achieved from bacterial CPS vaccines. Using their synthetic approach, affinity chromatography matrix could also be provided, thus allowing isolation and quantitation of antibodies. Importantly, they suggested that the patients’ own purified antibodies would be useful therapeutics after either radiolabeling
or drug-conjugation. Such an approach sounds very appealing from a regulatory point of view since it would avoid dealing with ‘humanized’ mouse monoclonal antibodies. Automated solid-phase syntheses of some of the above carbohydrate antigens has also been achieved, thus further expanding the scope of cancer vaccines [41]. www.drugdiscoverytoday.com
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Links
Figure 4. Idealized fully synthetic carbohydrate-based vaccine containing several carbohydrate and immunogenic T-cell epitopes which would most likely be useful against cancer cells, family of bacteria or parasites. Suitable T-cell epitopes are short, usually 15-mer peptides, recognized by most human Major Histocompatibily Complexes (MHC Class II). To reach most individuals, more than one allele-dependent T-cell epitope is required. An example of almost universal human T-cell epitope is tetanus toxin fragment 830–843 [11–14].
Non-carbohydrate anti-carbohydrate-based vaccines Future vaccines It is not unlikely that future glycan-based vaccines would ironically not contain any carbohydrate structures. The reasons for this is that there is an increasing body of evidences suggesting that carbohydrate mimotopes can be generated out of peptide sequences. The origin for this relatively novel strategy is based on the many observations that anti-idiotypic antibodies, provided peptide sequences that were mimicking the structures of carbohydrates. There are now several cases at hand demonstrating that antibodies generated from carbohydrate peptide-mimics could recognize and bind to bacterial CPS [42]. Two such good examples were provided by Pinto et al. (http://www.sfu.ca/chemistry/faculty/pinto.htm) with Streptococcus Group A and Shigella flexneri [43–45]. These findings are valuable in that readily prepared peptides acting as carbohydrate mimics, once attached to universal T-cell peptide epitopes, should provide entirely synthetic vaccines. Fundamental understandings on how the human immune system handles carbohydrate antigens is of prime importance for the future design of simple, ideally totally synthetic, carbohydrate-based vaccines. One such research activity is being actively pursued by the group of Kihlberg et al. in Sweden ([email protected]) who is evaluating the paramaters that could ultimately lead to rheumatoid arthritis vaccines [46–48].
Conclusions and comparisons of technology The rapid growth of sophisticated analytical tools, purification instruments, and proteo/glyco-mics allows the structural 334
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Sloan-Kettering Institute: carbohydrate-based antitumor vaccines: http://www.mskcc.org/mskcc/html/11263.cfm The development of anti-cancer carbohydrate vaccines: http:// www.nd.edu/aostafin/benl/2000/page5.html http://pubs.acs.org/isubscribe/journals/cen/78/i35/html/ 7835notw8.html Neisseria Meningitidis, mode of infection: http://www.brown.edu/ Courses/Bio_160/Projects1999/bmenin/nmenin.html Optimer Programmed One-Pot Synthesis Technology-OPopS(TM) Technology: http://www.optimerpharma.com/core_technology.htm Phase III Theratope1 vaccine trial: http://www.biomira.com/news/ detailNewsRelease/166/%3E PADRE carrier/adjuvant: http://www.epimmune.com/technology/ padre.cfm Aventis MenactraTM vaccine for protection against meningococcal infection. First candidate quadrivalent conjugate meningococcal vaccine: http://www.aventispasteur.com/index.cfm?FA=25022004
indentifications of an increasing number of target carbohydrate antigens of microbial origins and disease states. When coupled to our rising arsenals of chemo-enzymatic skills to build complex carbohydrate structures, including CPS, and our understanding of immune processing to such carbohydrate antigens, it is likely that we will witness more and better carbohydrate-based vaccines. It makes no doubt that critical milestones reside in recent progress achieved in synthetic carbohydrate and organic chemistry. Modern, stepwise and multistep syntheses of complex carbohydrates, although efficient in providing clean compounds, are usually to cumbersome for practical and largescale applications. Hence, the progress made by the one-pot solution-phase or solid-phase syntheses are likely to surpass more classical approaches. The above strategies, when coupled to well-established solid-phase peptide syntheses of immunogenic T-cell epitopes, are amenable to scale-up processes that will generate large amount of the small molecular weight candidate vaccines. As discussed above, fully synthetic vaccines are easier to characterized, handle, and are thus more likely to be rapidly approved by health organisms such as the Food and Drug Administration (FDA).
Related articles Danishefsky, S.J. and Allen, J.R. (2000) From the laboratory to the clinic: a retrospective on fully synthetic carbohydrate-based anticancer vaccines. Angew. Chem. Int. Ed. 39. 836–863 Borman, S. (2004) Carbohydrate vaccines. C&EN August 9, 31–34 (http:// pubs.acs.org/cen/coverstory/8232/8232vaccines.html) Borman, S. (2002) Vaccines by automated synthesis. C&EN January 21, 43–44 (http://pubs.acs.org/isubscribe/journals/cen/80/i03/html/ 8003sci4.html) Keding, S.J. and Danishefsky, S.J. (2003) Synthetic carbohydrate-based vaccines. Carbohydr. Based Drug Discov. 1, 381–406
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Not withstanding the recent success encountered with the semi-synthetic Hib vaccine, the complexity of bacterial polysaccharides are not always amenable to similar one-pot oligomerization. In the above case, the simple repeating units were linked together by a one-step, solution-phase phosphodiester assembly, similarly to those observed in oligonucleotides but not widely distributed. The situation could be even more complicated when several complex repeating units, se pentasaccharides, are required to form a conformational epitope that is reached when only half a dozen such structures are assembled. It is our belief that the ideal vaccines of the future will be constituted of synthetic carbohydrate/peptide antigens assembled on an equally synthetic small immunogenic peptides. Because of the complexity of the human T-cells, it is also likely that several T-cell peptide epitopes would be required to mount a universal vaccine. As vaccination for infants in developing countries is not a simple task, multivalent vaccines or cocktail of vaccines is highly recommended. The structure of an idealized vaccine is illustrated in Fig. 4. Equally important could be the combination of various antigens into liposomes for which the immunoadjuvant properties have long been recognized [49,50].
Outstanding issues Are productions of synthetic bacterial capsular polysaccharides cost effective compared to isolation from bacterial cultures? Are governmental health regulations more likely to accept synthetic vaccines in comparison to those obtained from bacteria? Are their truly universal T-cell epitopes? Totally synthetic vaccines, myth or reality?
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