Indian J Med Res 119 (Suppl) May 2004, pp 7-12
Short-chain oligosaccharide protein conjugates as experimental pneumococcal vaccines W.T.M. Jansen & Harm Snippe Eijkman-Winkler Institute, Heidelberglaan 100, 3584CX Utrecht, The Netherlands Received August 7, 2003 Streptococcus pneumoniae remains a major cause of acute respiratory infections worldwide and is responsible for approximately 1 million childhood deaths each year. Despite the widespread use of antibiotics, the mortality and morbidity of pneumococcal disease remains high. Therefore, effective vaccines to prevent pneumococcal disease are needed. Bacterial vaccine development in general follows a similar track starting from large, rather crude vaccines (whole live, attenuated pathogen) towards smaller, better defined subunit vaccines. Ultimately, this track leads to the development and evaluation of minimal, highly defined subunit vaccines, based on a collection of single protective epitopes. This mini-review deals with capsular saccharide based vaccines. After a short overview of the development of pneumococcal vaccines from the 23 - valent polysaccharide vaccines to polysaccharide-protein conjugate vaccines, it focuses on the vaccine potential of synthetic oligosaccharides, conjugated to carrier proteins. Key words Oligosaccharide - Streptococcus pneumoniae - vaccine
Worldwide, Streptococcus pneumoniae remains a major cause of acute respiratory bacterial infections, leading to approximately 1 million childhood deaths each year 1. Antibiotic resistance is a growing problem in the treatment of pneumococcal infections2. Despite the wide spread use of antibiotics, the mortality and morbidity of pneumococcal disease remains high3. In addition, resistant pneumococci are increasingly observed3-5. Recently, a report describing vancomycin tolerant pneumococci has alarmed the scientific community6. The main risk groups for pneumococcal disease are infants below 2 yr and elderly above 60 yr 7. Worldwide, approximately 1 million children die each year from pneumococcal pneumonia8. Therefore, renewed efforts have been undertaken to develop effective vaccines against pneumococci.
polysaccharide (PS) 9. Pneumococcal capsular types differ significantly in their virulence10. Of the 90 serotypes known today, only a limited number of 20 serotypes cause the majority (90%) of disease10. Serotypes most commonly isolated from patients include serotypes 1 and 14 from the blood, serotypes 6, 10 and 23 from the cerebrospinal fluid and serotypes 3, 19 and 23 from the middle ear fluid of infants11. Serotypes 6A, 6B, 14, 19F and 23F are the main paediatric serotypes9. These serotypes are poorly immunogenic, which might be the reason for their success as a pathogen in infants. The capsular PS are very long polymers of either linear or branched repeating units consisting of 2 (serotypes 3, 37) to 8 (serotype 17A) monosaccharides10. Host protection against Streptococcus pneumoniae is mainly mediated by complement and antibody dependent phagocytosis. Capsular PS are the most important virulence factors of pneumococci9. Capsular PS provide resistance against complement mediated
Capsular polysaccharide Pneumococci can be subdivided into more than 90 different serotypes, based on their capsular 7
8
INDIAN J MED RES (SUPPL) MAY 2004
phagocytosis12 and shield the inner structures of the pneumococcus from the immune system 13 . As a consequence, only antibodies against the capsular PS have been shown to fully protect humans against invasive pneumococcal disease. However, PS are so called thymus independent antigens, which means that they do not recrute a T helper cell response14. As a consequence, the antibody response is mainly restricted to IgM and IgA, affinity maturation does not occur, and B memory cells are not produced14. 23-valent PS vaccine The capsular PS of the 23 most prevalent serotypes have been included in the licenced 23-valent PS vaccine15,16. This vaccine provides protection against 73 per cent of the strains, and reduces the risk of systemic infection in the adult population to 83 per cent17. The efficacy of the vaccine, however is debatable in groups at risk for pneumococcal infections18,19. In the elderly, ineffective antibody responses after vaccination have been reported. However, a meta analysis of clinical PS vaccination studies including more than 65000 patients, did not show differences in vaccine induced antibody levels between adults and elderly17. Nevertheless, in the elderly, avidity of these antibodies is lower, which might correlate with an increased vulnerability to pneumococcal disease 20 . For infants, the poor immunogenicity of the PS vaccine is beyond doubt21,22 and thereby is unsuitable. Polysaccharide-protein conjugate vaccines The conventional 23-valent PS vaccine provides insufficient protection in groups at risk for pneumococcal infection. To overcome the thymus independency of PS antigens, PS are conjugated to carrier proteins, according to the immunological concepts developed in the mid seventies. These conjugate vaccines are immunogenic in infants, but in general, antibody responses in adults are not improved as compared to the conventional 23valent PS vaccine23. Recently clinical phase III trials have been conducted for several conjugate vaccines24. These vaccines include the serotypes 4, 6B, 9V, 14, 18C and 23F. As carrier proteins, either diphteria toxoid CRM197, meningococcus B outer membrane proteins or tetanus toxoid are used25. Several of these conjugate vaccines have been licenced and seem effective against
pneumococcal pneumoniae24, but to a lesser extend to otitis media26. It is therefore tempting to assume that the major obstacles in the prevention of pneumococcal infections have been overcome. Despite the clinical and commercial potential of these vaccines, a number of major problems can still be envisioned. Protective capacities of these vaccines have not been fully established yet, since information about protective opsonic antibody levels in humans is still lacking. In addition, the immunogenicity varies among the serotypes. Especially serotype 6B may be a problematic serotype in currently tested conjugate vaccines27. Furthermore, multivalent conjugate vaccines are very expensive and therefore not appropriate for vaccination programmes in the developing world. The most important problem is the number of serotypes that has to be included in the vaccine. Although a multivalent conjugate vaccine that comprises the 9 different most virulent serotypes would cover 80 to 90 per cent of the pneumococcal serotypes isolated worldwide from patients11, it is to be expected that a shift in incidence towards non-vaccine serotypes may occur, or new serotypes may emerge. Since the 23-valent PS vaccine has proven to be safe, and the immunogenicity of each serotype seems not to be suppressed by the other serotypes, similar benefits can be expected from multivalent PS conjugate vaccines. Nevertheless, the efficacy and safety of the vaccine should be controlled when additional serotypes have to be incorporated in the vaccine. Immunogenicity may be hampered by antigenic competition, carrier suppression28, unwanted cross reactions with host antigens may occur29 and finally, since capsular PS are indirectly linked to cell wall-PS30, increasing the number or doses of serotypes in the vaccine, will result in a similar increase of cell wall-PS content, which may affect the safety of the vaccine. Protein vaccines Taken into account these obstacles towards a safe and effective, worldwide pneumococcal vaccine, other vaccine strategies should also be considered. To overcome the disadvantages of serotype-specific vaccines, a number of pneumococcal proteins are being evaluated as vaccine candidates. These proteins include pneumolysin, autolysin, pneumococcal surface protein A (PspA), and pneumococcal surface adhesin A (PsaA). Although most proteins induce a certain level of protection
JANSEN & SNIPPE : PNEUMOCOCCAL VACCINES
against pneumococcal carriage and invasive disease in mice, their protective capacities in humans remain uncertain31,32.
9
saccharides induced opsonic anti PS antibodies in mouse models and rabbit models33,39-43. For all serotypes tested so far, anti-PS responses could be induced by very small OS fragments comprising only 1 repeating unit or less of the corresponding PS (Table). The serotypes listed in the Table belong to the most common ones isolated from adults and infants. Immune responses against the OS were highly specific and interestingly, some of the OSprotein conjugate vaccines even induced higher levels of anti-PS antibodies than the conventional PS-protein conjugate counterpart41.
Synthetic oligosaccharides in pneumococcal vaccines Pneumococcal capsular PS epitopes can be presented by synthetic oligosaccharides (OS). The synthetic components can be conjugated to a carrier protein with conventional coupling chemistry, to obtain semi-synthetic conjugate vaccines. The preparation of synthetic saccharides that represent fragments of pneumococcal PS antigens is traditionally difficult. Nevertheless, the combination of organic synthesis and biosynthesis using glycosyl transferases has lead to the production of numerous synthetic saccharides for serotypes 1-4, 6A/B, 7F, 8, 9A/V, 14, 17F, 18C, 19A/F, 22F, 23F, 27 and 2933. These saccharides have been used as inhibitors to determine the epitope specificity of anti-PS antibodies. In addition, synthetic OS can be used to study, and block, pneumococcal adhesion to epithelial cells34-38. Finally, coupled to carrier proteins, a growing number of synthetic saccharides is evaluated as a conjugate vaccine in murine models. Within the vaccine department at the University Medical Center, Utrecht in collaboration with the Bijvoet Centre in Utrecht, several synthetic saccharides are under investigation. Synthetic 6B, 17F and 23F
Although the vaccine potential of small synthetic OS is clear from studies in murine models, their potential as vaccine candidates in humans is still uncertain. It is known that epitope size on pneumococcal PS can vary between serotypes42,52, between species42 and with age53. In contrast to mice, humans may require longer saccharides for an optimal immune response, presumably because of the better presentation of conformational epitopes53. Again, this may vary per serotype since 6A and 6B41, but not 23F42 OS fragments were recognized by human vaccination antisera, indicating the presence of epitopes for human anti-PS antibodies on the former but not the latter OS fragments. A potential drawback of minimal synthetic OS conjugate vaccines is the absence of displaying
Table. Minimal oligosaccharide (OS) fragments conjugated to carrier proteins induced (protective) anti-PS responses in mice and rabbits OS chain lengtha inducing (protective) anti-PS responses Serotype
Carrierprotein
mice Anti- PS responseb
a
References
rabbits protectionc
Anti-PS response
protectiond
3
CRM 197
di, tri, tetra
di, tri, tetra
ND
ND
40,43,44
6A
KLH
tri, tetra
ND
tri, tetra
tri, tetra
41,45-47
6B
KLH
di, tri, tetra
tetra
di, tri, tetra
di, tri, tetra
14
CRM 197
tetra, octa
ND
ND
ND
39,48
17F
KLH
di, tri
di, tri
di, tri
ND
44,49,50
23F
KLH
ND
ND
tetra
ND
42,51
, number of monosaccharides within 1 OS fragment, coupled to a carrier protein; b, as measured by ELISA using PS as coating antigen41; , measured in a mouse challenge model41; d, passive protection of mice by anti OS-antibodies raised in rabbits41; ND, not done
c
10
INDIAN J MED RES (SUPPL) MAY 2004
conformational epitopes, which are present on the whole PS. On the other hand, the thymus dependency of an OS-conjugate increases with shortening of the OS chain length. Therefore, for PS containing conformational epitopes, an optimum OS length has to be defined, long enough to express conformational epitopes, yet short enough to induce an optimal Th cell response. To obtain OS that contain several repeating units of the PS, three methods can be applied: partial hydrolysis of the PS, chemical synthesis, or biosynthesis33. Several OS have been successfully prepared by hydrolysis. A disadvantage of this method is that the production of these OS is less well controlled leading to less defined products (size and exact bound hydrolized) and the presence of impurities. Chemical synthesis of OS of several repeating units starts to become possible, but is still very complex, requiring a large number of synthesis steps. The combination of chemical synthesis and biosynthesis might be a promising alternative in the near future to produce large OS at sufficient amounts. Information about the biosynthetic pathways of the capsular PS of serotypes 3, 14 and 19F has become available33. Several glycosyl transferases are identified and cloned 54-56 and one of them has already been produced at a large scale57. Even conjugation of certain saccharides to a carrier protein or peptide might be performed in part enzymatically in the future, since several enzymes have now been identified that can glycosylate specific amino acids residues 54,58. In addition to new strategies to synthesize saccharides, recently developed techniques such as Matrix-Associated Laser Desorption time of flight mass spectrometry (MALDItof) facilitate and improve the characterization of the product. Our findings and the work of others on synthetic OSprotein conjugates encourage the exploration of synthetic OS conjugate vaccines as a pneumococcal vaccine. Nevertheless, to establish whether animals and humans can produce antibodies against minimal synthetic OS, is only the first step towards a synthetic pneumococcal vaccine. Next step is to define the most immunogenic, protective epitopes on PS and optimize the presentation of such epitopes to the immune system. In conclusion, considering the large number of serotypes, the generation of new synthetic saccharides should be stimulated. Depending on the serotype, when
needed and when possible, natural PS may then be replaced sequentially by their (bio)synthetic OS counterparts in future semi-synthetic pneumococcal vaccines. References 1.
Pneumococcal vaccines. WHO position paper. Wkly Epidemiol Rec 1999; 74 : 177-83.
2.
Tan TQ. Pneumococcal conjugate vaccines - Implications for community antibiotic prescribing. Curr Opin Microbiol 2000; 3 : 502-7.
3.
Kellner JD. Drug-resistant Streptococcus pneumoniae infections: clinical importance, drug treatment, and prevention. Semin Respir Infect 2001; 16 : 186-95.
4.
Whitney CG, Farley MM, Hadler J, Harrison LH, Lexau C, Reingold A, et al. Increasing prevalence of multidrug-resistant Streptococcus pneumoniae in the United States. N Engl J Med 2000; 343 : 1917-24.
5.
Schreiber JR, Jacobs MR. Antibiotic-resistant pneumococci. Pediatr Clin North Am 1995; 42 : 519-37.
6.
Wenzel RP, Edmond MB. Managing antibiotic resistance. N Engl J Med 2000; 343 : 1961-3.
7.
Rijkers GT, Sanders LA, Zegers BJ. Anti-capsular polysaccharide antibody deficiency states. Immunodeficiency 1993; 5 : 1-21.
8.
Sniadack DH, Schwartz B, Lipman H, Bogaerts J, Butler JC, Dagan R, et al. Geographic and temporal differences in serotype and serogroup distribution of sterile site pneumococcal isolates from children - Implications for vaccine strategies. Pediatr Infect Dis J 1995; 14 : 503-10.
9.
AlonsoDeVelasco E, Verheul AF, Verhoef J, Snippe H. Streptococcus pneumoniae: Virulence factors, pathogenesis, and vaccines. Microbiol Rev 1995; 59 : 591-603.
10. van Dam JE, Fleer A, Snippe H. Immunogenicity and immunochemistry of Streptococcus pneumoniae capsular polysaccharides. Antonie Van Leeuwenhoek 1990; 58 : 1-47. 11. Hausdorff WP, Yothers G, Dagan R, Kilpi T, Pelton SI, Cohen R, et al. Multinational study of pneumococcal serotypes causing acute otitis media in children. Pediatr Infect Dis J 2002; 21 : 1008-16. 12. Brown EJ, Joiner KA, Cole RM, Berger M. Localization of complement component 3 on Streptococcus pneumoniae: anticapsular antibody causes complement deposition on the pneumococcal. Capsule Infect Immun 1983; 39 : 403-9. 13. Nielsen SV, Sorensen UB, Henrichsen J. Antibodies against pneumococcal C-polysaccharide are not protective. Microb Pathog 1993;14 : 299-305. 14. Stein KE. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J Infect Dis 1992; 165 (Suppl 1) : S49-S52.
JANSEN & SNIPPE : PNEUMOCOCCAL VACCINES 15. Requejo HI. Polyvalent pneumococcal polysaccharide vaccines; a review of the literature. Rev Hosp Clin Fac Med Sao Paulo 1993; 48 : 130-8. 16. Robbins JB, Austrian R, Lee CJ, Rastogi SC, Schiffman G, Henrichsen J, et al. Considerations for formulating the secondgeneration pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups. J Infect Dis 1983; 148 : 1136-59. 17. Hutchison BG, Oxman AD, Shannon HS, Lloyd S, Altmayer CA, Thomas K. Clinical effectiveness of pneumococcal vaccine. Meta-analysis. Can Fam Physician 1999; 45 : 2381-93. 18. Avanzini MA, Carra AM, Maccario R, Zecca M, Pignatti P, Marconi M, et al. Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation. J Clin Immunol 1995; 15 : 137-44. 19. King JC Jr, Vink PE, Farley JJ, Parks M, Smilie M, Madore D, et al. Comparison of the safety and immunogenicity of a pneumococcal conjugate with a licensed polysaccharide vaccine in human immunodeficiency virus and non-human immunodeficiency virus-infected children. Pediatr Infect Dis J 1996; 15 : 192-6. 20. Romero-Steiner S, Musher DM, Cetron MS, Pais LB, Groover JE, Fiore AE, et al. Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity. Clin Infect Dis 1999; 29 : 281-8. 21. Meyer M, Gahr M. Immunological principles of polysaccharideprotein conjugate vaccination. Monatsschr Kinderheilkd 1993; 141 : 770-6. 22. Pomat WS, Lehmann D, Sanders RC, Lewis DJ, Wilson J, Rogers S, et al. Immunoglobulin G antibody responses to polyvalent pneumococcal vaccine in children in the highlands of Papua New Guinea. Infect Immun 1994; 62 : 1848-53. 23. Briles DE, Ades E, Paton JC, Sampson JS, Carlone GM, Huebner RC, et al. Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae. Infect Immun 2000; 68 : 796-800. 24. Lee LH, Lee CJ, Frasch CE. Development and evaluation of pneumococcal conjugate vaccines: clinical trials and control tests. Crit Rev Microbiol 2002; 28 : 27-41. 25. Eskola J. Polysaccharide-based pneumococcal vaccines in the prevention of acute otitis media. Vaccine 2000; 19 (Suppl 1) : S78-S82.
11
28. Dagan R, Eskola J, Leclerc C, Leroy O. Reduced response to multiple vaccines sharing common protein epitopes that are administered simultaneously to infants. Infect Immun 1998; 66 : 2093-8. 29. Finne J, Leinonen M, Makela PH. Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 1983; 2 : 355-7. 30. Sorensen UB, Henrichsen J, Chen HC, Szu SC. Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. Microb Pathog 1990; 8 : 325-34. 31. Briles DE, Hollingshead SK, Nabors GS, Paton JC, BrooksWalter A. The potential for using protein vaccines to protect against otitis media caused by Streptococcus pneumoniae. Vaccine 2000;19 (Suppl 1) : S87-S95. 32. Zysk G, Bongaerts RJ, ten Thoren E, Bethe G, Hakenbeck R, Heinz HP. Detection of 23 immunogenic pneumococcal proteins using convalescent phase serum. Infect Immun 2000; 68 : 3740-3. 33. Kamerling JP. Pneumococcal polysaccharides: A chemical view. In: Tomasz A, editor. Streptococcus pneumoniae, molecular biology & mechanisms of disease. New York: Mary Ann Liebert; 1999 p. 81-114. 34. Sharon N, Ofek I. Safe as mother’s milk: Carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconj J 2000; 17 : 659-64. 35. Ukkonen P, Varis K, Jernfors M, Herva E, Jokinen J, Ruokokoski E, et al. Treatment of acute otitis media with an antiadhesive oligosaccharide: a randomised, double-blind, placebo-controlled trial. Lancet 2000; 356 : 1398-402. 36. Tong HH, McIver MA, Fisher LM, DeMaria TF. Effect of lacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adherence of otitis media-associated serotypes of Streptococcus pneumoniae to chinchilla tracheal epithelium. Microb Pathog 1999; 26 : 111-9. 37. Barthelson R, Mobasseri A, Zopf D, Simon P. Adherence of Streptococcus pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides. Infect Immun 1998; 66 : 1439-44. 38. Idanpaan-Heikkila I, Simon PM, Zopf D, Vullo T, Cahill P, Sokol K, et al. Oligosaccharides interfere with the establishment and progression of experimental pneumococcal pneumonia. J Infect Dis 1997; 176 : 704-12.
26. Straetemans M, Sanders EA, Veenhoven RH, Schilder AG, Damoiseaux RA, Zielhuis GA. Pneumococcal vaccines for preventing otitis media. Cochrane Database Syst Rev 2002; 2 : CD001480.
39. Mawas F, Niggemann J, Jones C, Corbel MJ, Kamerling JP, Vliegenthart JF. Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197. Infect Immun 2002; 70 : 5107-14.
27. Sorensen RU, Leiva LE, Giangrosso PA, Butler B, Javier FC III, Sacerdote DM, et al. Response to a heptavalent conjugate Streptococcus pneumoniae vaccine in children with recurrent infections who are unresponsive to the polysaccharide vaccine. Pediatr Infect Dis J 1998; 17 : 685-91.
40. Benaissa-Trouw B, Lefeber DJ, Kamerling JP, Vliegenthart JF, Kraaijeveld K, Snippe, H. Synthetic polysaccharide type 3related di-, tri-, and tetrasaccharide- CRM(197) conjugates induce protection against Streptococcus pneumoniae type 3 in mice. Infect Immun 2001; 69 : 4698-701.
12
INDIAN J MED RES (SUPPL) MAY 2004
41. Jansen WT, Hogenboom S, Thijssen MJ, Kamerling JP, Vliegenthart JF, Verhoef J, et al. Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice. Infect Immun 2001; 69 : 787-93. 42. Alonso De V, Verheul AF, van Steijn AM, Dekker HA, Feldman RG, Fernandez IM, et al. Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide- and native polysaccharide-protein conjugate vaccines: Comparison with human anti-polysaccharide 23F IgG. Infect Immun 1994; 62 : 799-808. 43. Snippe H, van Dam JE, Van Houte AJ, Willers JM, Kamerling JP, Vliegenthart JF. Preparation of a semisynthetic vaccine to Streptococcus pneumoniae Type 3. Infect Immun 1983; 42 : 842-4. 44. Lefeber DJ, Kamerling JP, Vliegenthart JF. Synthesis of Streptococcus pneumoniae type 3 neoglycoproteins varying in oligosaccharide chain length, loading and carrier protein. Chemistry 2001; 7 : 4411-21. 45. Thijssen MJ, Bijkerk MH, Kamerling JP, Vliegenthart JF. Synthesis of four spacer-containing tetrasaccharides' that represent four possible repeating units of the capsular polysaccharide of Streptococcus pneumoniae type 6B. Carbohydr Res 1998; 306 : 111-25. 46. Thijssen MJ, van Rijswijk MN, Kamerling JP, Vliegenthart JF. Synthesis of spacer-containing di- and tri-saccharides that represent parts of the capsular polysaccharide of Streptococcus pneumoniae type 6B. Carbohydr Res 1998; 306 : 93-109.
synthetic di-, tri- and tetrasaccharide conjugates in mice and rabbits. Vaccine 2001; 20 : 19-21. 50. Jones C, Whitley C, Lemercinier X. Full assignment of the proton and carbon NMR spectra and revised structure for the capsular polysaccharide from Streptococcus pneumoniae type 17F. Carbohydr Res 2000; 325 : 192-201. 51. van Steijn AM, Kamerling JP, Vliegenthart JF. Synthesis of a spacer-containing repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 23F. Carbohydr Res 1991; 211 : 261-77. 52. Alonso De V, Verheul AF, Veeneman GH, Gomes LJ, van Boom JH, Verhoef J, et al. Protein-conjugated synthetic di- and trisaccharides of pneumococcal type 17F exhibit a different immunogenicity and antigenicity than tetrasaccharide. Vaccine 1993;11 : 1429-36. 53. Pichichero ME, Porcelli S, Treanor J, Anderson P. Serum antibody responses of weaning mice and two-year-old children to pneumococcal-type 6A protein conjugate vaccines of differing saccharide chain lengths. Vaccine 1998; 16 : 83-91. 54. Shpak E, Leykam JF, Kieliszewski MJ. Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-Oglycosylation codes. Proc Natl Acad Sci USA 1999; 96 : 1473641. 55. Di Virgilio S, Glushka J, Moremen K, Pierce M. Enzymatic synthesis of natural and 13C enriched linear poly-Nacetyllactosamines as ligands for galectin-1. Glycobiology 1999; 9 : 353-64.
47. Thijssen MJ, Halkes KM, Kamerling JP, Vliegenthart JF. Synthesis of a spacer-containing tetrasaccharide representing a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 6B. Bioorg Med Chem 1994; 2 : 1309-17.
56. Elling L. Glycobiotechnology: enzymes for the synthesis of nucleotide sugars. Adv Biochem Eng Biotechnol 1997; 58 : 89144.
48. Niggemann J, Kamerling JP, Vliegenthart JF. Beta-1,4galactosyltransferase-catalyzed synthesis of the branched tetrasaccharide repeating unit of Streptococcus pneumoniae type 14. Bioorg Med Chem 1998; 6 : 1605-12.
57. Yamamoto T, Nagae H, Kajihara Y, Terada I. Mass production of bacterial alpha 2,6-sialyltransferase and enzymatic syntheses of sialyloligosaccharides. Biosci Biotechnol Biochem 1998; 62 : 210-4.
49. Jansen WT, Verheul AF, Veeneman GH, van Boom JH, Snippe H. Revised interpretation of the immunological results obtained with pneumococcal polysaccharide 17F derived
58. Imperiali B, O’Connor SE. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 1999; 3 : 643-9.
Reprint requests: Dr W.T.M. Jansen, Eijkman-Winkler Institute, G04-614 UMC, Microbiology Heidelberglaan 100 3584 CX Utrecht, The Netherlands e-mail:
[email protected]
