Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jun;65(6):2088–2093. doi: 10.1128/iai.65.6.2088-2093.1997

Synthesis and immunological properties of Vi and di-O-acetyl pectin protein conjugates with adipic acid dihydrazide as the linker.

Z Kossaczka 1, S Bystricky 1, D A Bryla 1, J Shiloach 1, J B Robbins 1, S C Szu 1
PMCID: PMC175288  PMID: 9169736

Abstract

The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals > or = 5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-alpha(1-->4)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 microg, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5- to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast beta-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.

Full Text

The Full Text of this article is available as a PDF (488.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acharya I. L., Lowe C. U., Thapa R., Gurubacharya V. L., Shrestha M. B., Cadoz M., Schulz D., Armand J., Bryla D. A., Trollfors B. Prevention of typhoid fever in Nepal with the Vi capsular polysaccharide of Salmonella typhi. A preliminary report. N Engl J Med. 1987 Oct 29;317(18):1101–1104. doi: 10.1056/NEJM198710293171801. [DOI] [PubMed] [Google Scholar]
  2. Anderson P. W., Pichichero M. E., Insel R. A., Betts R., Eby R., Smith D. H. Vaccines consisting of periodate-cleaved oligosaccharides from the capsule of Haemophilus influenzae type b coupled to a protein carrier: structural and temporal requirements for priming in the human infant. J Immunol. 1986 Aug 15;137(4):1181–1186. [PubMed] [Google Scholar]
  3. Anderson P., Pichichero M. E., Insel R. A. Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197. J Clin Invest. 1985 Jul;76(1):52–59. doi: 10.1172/JCI111976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bystricky S., Szu S. C. O-acetylation affects the binding properties of the carboxyl groups on the Vi bacterial polysaccharide. Biophys Chem. 1994 Jul;51(1):1–7. doi: 10.1016/0301-4622(94)00002-6. [DOI] [PubMed] [Google Scholar]
  6. Chihara G., Maeda Y. Y., Hamuro J. Current status and perspectives of immunomodulators of microbial origin. Int J Tissue React. 1982;4(3):207–225. [PubMed] [Google Scholar]
  7. Chu C., Schneerson R., Robbins J. B., Rastogi S. C. Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates. Infect Immun. 1983 Apr;40(1):245–256. doi: 10.1128/iai.40.1.245-256.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devi S. J., Robbins J. B., Schneerson R. Antibodies to poly[(2----8)-alpha-N-acetylneuraminic acid] and poly[(2----9)-alpha-N-acetylneuraminic acid] are elicited by immunization of mice with Escherichia coli K92 conjugates: potential vaccines for groups B and C meningococci and E. coli K1. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7175–7179. doi: 10.1073/pnas.88.16.7175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fattom A., Shiloach J., Bryla D., Fitzgerald D., Pastan I., Karakawa W. W., Robbins J. B., Schneerson R. Comparative immunogenicity of conjugates composed of the Staphylococcus aureus type 8 capsular polysaccharide bound to carrier proteins by adipic acid dihydrazide or N-succinimidyl-3-(2-pyridyldithio)propionate. Infect Immun. 1992 Feb;60(2):584–589. doi: 10.1128/iai.60.2.584-589.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HESTRIN S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J Biol Chem. 1949 Aug;180(1):249–261. [PubMed] [Google Scholar]
  11. Hoare D. G., Koshland D. E., Jr A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J Biol Chem. 1967 May 25;242(10):2447–2453. [PubMed] [Google Scholar]
  12. Inman J. K., Dintzis H. M. The derivatization of cross-linked polyacrylamide beads. Controlled introduction of functional groups for the preparation of special-purpose, biochemical adsorbents. Biochemistry. 1969 Oct;8(10):4074–4082. doi: 10.1021/bi00838a026. [DOI] [PubMed] [Google Scholar]
  13. Keitel W. A., Bond N. L., Zahradnik J. M., Cramton T. A., Robbins J. B. Clinical and serological responses following primary and booster immunization with Salmonella typhi Vi capsular polysaccharide vaccines. Vaccine. 1994;12(3):195–199. doi: 10.1016/0264-410x(94)90194-5. [DOI] [PubMed] [Google Scholar]
  14. Klugman K. P., Gilbertson I. T., Koornhof H. J., Robbins J. B., Schneerson R., Schulz D., Cadoz M., Armand J. Protective activity of Vi capsular polysaccharide vaccine against typhoid fever. Lancet. 1987 Nov 21;2(8569):1165–1169. doi: 10.1016/s0140-6736(87)91316-x. [DOI] [PubMed] [Google Scholar]
  15. LANDY M. Studies on Vi antigen. VI. Immunization of human beings with purified Vi antigen. Am J Hyg. 1954 Jul;60(1):52–62. doi: 10.1093/oxfordjournals.aje.a119703. [DOI] [PubMed] [Google Scholar]
  16. LANDY M. Studies on Vi antigen. VII. Characteristics of the immune response in the mouse. Am J Hyg. 1957 Jan;65(1):81–93. doi: 10.1093/oxfordjournals.aje.a119858. [DOI] [PubMed] [Google Scholar]
  17. Liao J., Nickerson K. G., Bystricky S., Robbins J. B., Schneerson R., Szu S. C., Kabat E. A. Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi. Infect Immun. 1995 Nov;63(11):4429–4432. doi: 10.1128/iai.63.11.4429-4432.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Peeters C. C., Evenberg D., Hoogerhout P., Käyhty H., Saarinen L., van Boeckel C. A., van der Marel G. A., van Boom J. H., Poolman J. T. Synthetic trimer and tetramer of 3-beta-D-ribose-(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. 1992 May;60(5):1826–1833. doi: 10.1128/iai.60.5.1826-1833.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peeters C. C., Tenbergen-Meekes A. M., Evenberg D. E., Poolman J. T., Zegers B. J., Rijkers G. T. A comparative study of the immunogenicity of pneumococcal type 4 polysaccharide and oligosaccharide tetanus toxoid conjugates in adult mice. J Immunol. 1991 Jun 15;146(12):4308–4314. [PubMed] [Google Scholar]
  20. Plotkin S. A., Bouveret-Le Cam N. A new typhoid vaccine composed of the Vi capsular polysaccharide. Arch Intern Med. 1995 Nov 27;155(21):2293–2299. [PubMed] [Google Scholar]
  21. Polotsky V. Y., Robbins J. B., Bryla D., Schneerson R. Comparison of conjugates composed of lipopolysaccharide from Shigella flexneri type 2a detoxified by two methods and bound to tetanus toxoid. Infect Immun. 1994 Jan;62(1):210–214. doi: 10.1128/iai.62.1.210-214.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pospísil M., Sandula J., Pipalová I., Hofer M., Viklická S. Hemopoiesis stimulating and radioprotective effects of carboxymethylglucan. Physiol Res. 1991;40(4):377–380. [PubMed] [Google Scholar]
  23. Robbins J. B., Chu C., Schneerson R. Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharide of their lipopolysaccharides. Clin Infect Dis. 1992 Aug;15(2):346–361. doi: 10.1093/clinids/15.2.346. [DOI] [PubMed] [Google Scholar]
  24. Robbins J. B., Schneerson R., Szu S. C. Perspective: hypothesis: serum IgG antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum. J Infect Dis. 1995 Jun;171(6):1387–1398. doi: 10.1093/infdis/171.6.1387. [DOI] [PubMed] [Google Scholar]
  25. Robbins J. D., Robbins J. B. Reexamination of the protective role of the capsular polysaccharide (Vi antigen) of Salmonella typhi. J Infect Dis. 1984 Sep;150(3):436–449. doi: 10.1093/infdis/150.3.436. [DOI] [PubMed] [Google Scholar]
  26. Schneerson R., Barrera O., Sutton A., Robbins J. B. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med. 1980 Aug 1;152(2):361–376. doi: 10.1084/jem.152.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Steinhoff M. C., Edwards K., Keyserling H., Thoms M. L., Johnson C., Madore D., Hogerman D. A randomized comparison of three bivalent Streptococcus pneumoniae glycoprotein conjugate vaccines in young children: effect of polysaccharide size and linkage characteristics. Pediatr Infect Dis J. 1994 May;13(5):368–372. doi: 10.1097/00006454-199405000-00007. [DOI] [PubMed] [Google Scholar]
  28. Szewczyk B., Taylor A. Immunochemical properties of Vi antigen from Salmonella typhi Ty2: presence of two antigenic determinants. Infect Immun. 1980 Aug;29(2):539–544. doi: 10.1128/iai.29.2.539-544.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Szu S. C., Bystricky S., Hinojosa-Ahumada M., Egan W., Robbins J. B. Synthesis and some immunologic properties of an O-acetyl pectin [poly(1-->4)-alpha-D-GalpA]-protein conjugate as a vaccine for typhoid fever. Infect Immun. 1994 Dec;62(12):5545–5549. doi: 10.1128/iai.62.12.5545-5549.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Szu S. C., Li X. R., Schneerson R., Vickers J. H., Bryla D., Robbins J. B. Comparative immunogenicities of Vi polysaccharide-protein conjugates composed of cholera toxin or its B subunit as a carrier bound to high- or lower-molecular-weight Vi. Infect Immun. 1989 Dec;57(12):3823–3827. doi: 10.1128/iai.57.12.3823-3827.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Szu S. C., Li X. R., Stone A. L., Robbins J. B. Relation between structure and immunologic properties of the Vi capsular polysaccharide. Infect Immun. 1991 Dec;59(12):4555–4561. doi: 10.1128/iai.59.12.4555-4561.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Szu S. C., Stone A. L., Robbins J. D., Schneerson R., Robbins J. B. Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals. J Exp Med. 1987 Nov 1;166(5):1510–1524. doi: 10.1084/jem.166.5.1510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Szu S. C., Taylor D. N., Trofa A. C., Clements J. D., Shiloach J., Sadoff J. C., Bryla D. A., Robbins J. B. Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein conjugate vaccines. Infect Immun. 1994 Oct;62(10):4440–4444. doi: 10.1128/iai.62.10.4440-4444.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Szu S., Lee C. J., Carlo D., Henrichsen J. Immunochemical characterization of cross-reactivity of pneumococcal group 9 capsular polysaccharide types 9N, 9A, 9L, and 9V. Infect Immun. 1981 Jan;31(1):371–379. doi: 10.1128/iai.31.1.371-379.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wagnerová J., Lísková A., Cervenáková L., Trnovec T., Ferencík M. The immunoadjuvant effect of soluble glucan derivatives in mice. Folia Microbiol (Praha) 1991;36(2):198–204. doi: 10.1007/BF02814503. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES