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. 1980 Aug 1;152(2):361–376. doi: 10.1084/jem.152.2.361

Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates

PMCID: PMC2185954  PMID: 6967514

Abstract

A method is presented for covalently bonding Haemophilus influenzae type b capsular polysaccharide (HIB Ps) to several proteins. The method is efficient and relies upon the use of adipic dihydrazide as a spacer between the capsular polysaccharide and the carrier protein. In contrast to the poor immunogenicity of the purified HIB Ps in mice and rabbits, the HIB Ps-protein conjugates induced serum anti-type b antibodies having bactericidal activity at levels shown to be protective in humans when low doses were injected subcutaneously in a saline solution. The antibody response in mice was related to the dose of the conjugates, increased with the number of injections, and could be primed by the previous injection of the carrier protein. The HIB Ps- protein conjugates were immunogenic in three different mouse strains. The importance of the carrier molecule for the enhanced immunogenicity of the HIB Ps-protein conjugates was shown by the failure of HIB Ps hybrids prepared with either the homologous polysaccharide or pneumococcus type 3 polysaccharide to induce antibodie in mice. Rabbits injected with the HIB Ps-protein conjugates emulsified in Freund's adjuvant produced high levels of serum anti-type b antibodies which induced a bactericidal effect upon H. influenzae type b organisms. It is proposed that the HIB Ps component of the polysaccharide protein conjugates has been converted to a thymic-dependent immunogen. This method may be used to prepare protein-polysaccharide conjugates with HIB Ps and other polysaccharides to be considered for human use.

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Selected References

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  1. Ames B. N., Mccann J., Yamasaki E. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res. 1975 Dec;31(6):347–364. doi: 10.1016/0165-1161(75)90046-1. [DOI] [PubMed] [Google Scholar]
  2. Anderson P., Peter G., Johnston R. B., Jr, Wetterlow L. H., Smith D. H. Immunization of humans with polyribophosphate, the capsular antigen of Hemophilus influenzae, type b. J Clin Invest. 1972 Jan;51(1):39–44. doi: 10.1172/JCI106794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arakatsu Y., Ashwell G., Kabat E. A. Immunochemical studies on dextrans. V. Specificity and cross-reactivity with dextrans of the antibodies formed in rabbits to isomaltonic and isomaltotrionic acids coupled to bovine serum albumin. J Immunol. 1966 Dec;97(6):858–866. [PubMed] [Google Scholar]
  4. Avery O. T., Goebel W. F. CHEMO-IMMUNOLOGICAL STUDIES ON CONJUGATED CARBOHYDRATE-PROTEINS : II. IMMUNOLOGICAL SPECIFICITY OF SYNTHETIC SUGAR-PROTEIN ANTIGENS. J Exp Med. 1929 Sep 30;50(4):533–550. doi: 10.1084/jem.50.4.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  6. Baker P. J. Homeostatic control of antibody responses: a model based on the recognition of cell-associated antibody by regulatory T cells. Transplant Rev. 1975;26:3–20. doi: 10.1111/j.1600-065x.1975.tb00172.x. [DOI] [PubMed] [Google Scholar]
  7. Borgoño J. M., McLean A. A., Vella P. P., Woodhour A. F., Canepa I., Davidson W. L., Hilleman M. R. Vaccination and revaccination with polyvalent pneumococcal polysaccharide vaccines in adults and infants. Proc Soc Exp Biol Med. 1978 Jan;157(1):148–154. doi: 10.3181/00379727-157-40010. [DOI] [PubMed] [Google Scholar]
  8. Chen R. F. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967 Jan 25;242(2):173–181. [PubMed] [Google Scholar]
  9. Crisel R. M., Baker R. S., Dorman D. E. Capsular polymer of Haemophilus influenzae, type b. I. Structural characterization of the capsular polymer of strain Eagan. J Biol Chem. 1975 Jul 10;250(13):4926–4930. [PubMed] [Google Scholar]
  10. Geyer H., Stirm S., Himmelspach K. Immunochemical properties of oligosaccharide-protein conjugates with Klebsiella-K2 specificity. I. Specificity and crossreactivity of anti-conjugate versus anti-bacterial antibodies. Med Microbiol Immunol. 1979 Jan 24;165(4):271–288. doi: 10.1007/BF02152925. [DOI] [PubMed] [Google Scholar]
  11. Goebel W. F., Avery O. T. CHEMO-IMMUNOLOGICAL STUDIES ON CONJUGATED CARBOHYDRATE-PROTEINS : I. THE SYNTHESIS OFp-AMINOPHENOL beta-GLUCOSIDE, p-AMINOPHENOL beta-GALACTOSIDE, AND THEIR COUPLING WITH SERUM GLOBULIN. J Exp Med. 1929 Sep 30;50(4):521–531. doi: 10.1084/jem.50.4.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gotschlich E. C., Rey M., Etienne J., Sanborn W. R., Triau R., Cvjetanović B. The immunological responses observed in field studies in Africa with group A meningococcal vaccines. Prog Immunobiol Stand. 1971;5:485–491. [PubMed] [Google Scholar]
  13. Gotschlich E. C., Rey M., Triau R., Sparks K. J. Quantitative determination of the human immune response to immunization with meningococcal vaccines. J Clin Invest. 1972 Jan;51(1):89–96. doi: 10.1172/JCI106801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Handzel Z. T., Argaman M., Parke J. C., Jr, Schneerson R., Robbins J. B. Heteroimmunization to the capsular polysaccharide of Haemophilus influenzae type b induced by enteric cross-reacting bacteria. Infect Immun. 1975 May;11(5):1045–1052. doi: 10.1128/iai.11.5.1045-1052.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Jenkin C. R., Karnovsky M. L., Rowley D. Preparation of an artificial antigen and immunity to mouse typhoid. Immunology. 1967 Oct;13(4):361–372. [PMC free article] [PubMed] [Google Scholar]
  17. Jörbeck H. J., Svenson S. B., Lindberg A. A. Immunochemistry of Salmonella O-antigens: specificity of rabbit antibodies against the O-antigen 4 determinant elicited by whole bacteria and O-antigen 4 specific saccharide-protein conjugates. J Immunol. 1979 Sep;123(3):1376–1381. [PubMed] [Google Scholar]
  18. Katz D. H., Paul W. E., Goidl E. A., Benacerraf B. Carrier function in anti-hapten immune responses. I. Enhancement of primary and secondary anti-hapten antibody responses by carrier preimmunization. J Exp Med. 1970 Aug 1;132(2):261–282. doi: 10.1084/jem.132.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lerman S. P., Romano T. J., Mond J. J., Heidelberger M., Thorbecke G. J. Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide. Cell Immunol. 1975 Feb;15(2):321–335. doi: 10.1016/0008-8749(75)90011-8. [DOI] [PubMed] [Google Scholar]
  20. Lindberg A. A., Rosenberg L. T., Ljunggren A., Garegg P. J., Svensson S., Wallin N. H. Effect of synthetic disaccharide-protein conjugate as an immunogen in Salmonella infection in mice. Infect Immun. 1974 Sep;10(3):541–545. doi: 10.1128/iai.10.3.541-545.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mitchell G. F., Humphrey J. H., Wiliamson A. R. Inhibition of secondary anti-hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide. Eur J Immunol. 1972 Oct;2(5):460–467. doi: 10.1002/eji.1830020516. [DOI] [PubMed] [Google Scholar]
  22. Orskov F., Orskov I., Sutton A., Schneerson R., Lin W., Egan W., Hoff G. E., Robbins J. B. Form variation in Escherichia coli K1: determined by O-acetylation of the capsular polysaccharide. J Exp Med. 1979 Mar 1;149(3):669–685. doi: 10.1084/jem.149.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pappenheimer A. M., Jr, Uchida T., Harper A. A. An immunological study of the diphtheria toxin molecule. Immunochemistry. 1972 Sep;9(9):891–906. doi: 10.1016/0019-2791(72)90163-2. [DOI] [PubMed] [Google Scholar]
  24. Paul W. E., Katz D. H., Benacerraf B. Augmented anti-S 3 antibody responses to an S 3 -protein conjugate. J Immunol. 1971 Sep;107(3):685–688. [PubMed] [Google Scholar]
  25. Peltola H., Käyhty H., Sivonen A., Mäkelä H. Haemophilus influenzae type b capsular polysaccharide vaccine in children: a double-blind field study of 100,000 vaccinees 3 months to 5 years of age in Finland. Pediatrics. 1977 Nov;60(5):730–737. [PubMed] [Google Scholar]
  26. Pittman M. VARIATION AND TYPE SPECIFICITY IN THE BACTERIAL SPECIES HEMOPHILUS INFLUENZAE. J Exp Med. 1931 Mar 31;53(4):471–492. doi: 10.1084/jem.53.4.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rajewsky K., Schirrmacher V., Nase S., Jerne N. K. The requirement of more than one antigenic determinant for immunogenicity. J Exp Med. 1969 Jun 1;129(6):1131–1143. doi: 10.1084/jem.129.6.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robbins J. B., Parke J. C., Jr, Schneerson R., Whisnant J. K. Quantitative measurement of "natural" and immunization-induced Haemophilus influenzae type b capsular polysaccharide antibodies. Pediatr Res. 1973 Mar;7(3):103–110. doi: 10.1203/00006450-197303000-00001. [DOI] [PubMed] [Google Scholar]
  29. Robbins J. B. Vaccines for the prevention of encapsulated bacterial diseases: current status, problems and prospects for the future. Immunochemistry. 1978 Nov;15(10-11):839–854. doi: 10.1016/0161-5890(78)90117-7. [DOI] [PubMed] [Google Scholar]
  30. Schneerson R., Bradshaw M., Whisnant J. K., Myerowitz R. L., Parke J. C., Jr, Robbins J. B. An Escherichia coli antigen cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b: occurrence among known serotypes, and immunochemical and biologic properties of E. coli antisera toward H. influenzae type b. J Immunol. 1972 Jun;108(6):1551–1562. [PubMed] [Google Scholar]
  31. Schneerson R., Rodrigues L. P., Parke J. C., Jr, Robbins J. B. Immunity to disease caused by Hemophilus influenzae type b. II. Specificity and some biologic characteristics of "natural," infection-acquired, and immunization-induced antibodies to the capsular polysaccharide of Hemophilus influenzae type b. J Immunol. 1971 Oct;107(4):1081–1089. [PubMed] [Google Scholar]
  32. Smith D. H., Peter G., Ingram D. L., Harding A. L., Anderson P. Responses of children immunized with the capsular polysaccharide of Hemophilus influenzae, type b. Pediatrics. 1973 Nov;52(5):637–644. [PubMed] [Google Scholar]
  33. Sullivan B., Bonaventura J., Bonaventura C. Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2558–2562. doi: 10.1073/pnas.71.6.2558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Svenson S. B., Nurminen M., Lindberg A. A. Artificial Salmonella vaccines: O-antigenic oligosaccharide-protein conjugates induce protection against infection with Salmonella typhimurium. Infect Immun. 1979 Sep;25(3):863–872. doi: 10.1128/iai.25.3.863-872.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vann W. F., Jann K. Structure and serological specificity of the K13-antigenic polysaccharide (K13 antigen) of urinary tract-infective Escherichia coli. Infect Immun. 1979 Jul;25(1):85–92. doi: 10.1128/iai.25.1.85-92.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weller P. F., Smith A. L., Anderson P., Smith D. H. The role of encapsulation and host age in the clearance of Haemophilus influenzae bacteremia. J Infect Dis. 1977 Jan;135(1):34–41. doi: 10.1093/infdis/135.1.34. [DOI] [PubMed] [Google Scholar]
  37. Wong K. H., Barrera O., Sutton A., May J., Hochstein D. H., Robbins J. D., Robbins J. B., Parkman P. D., Seligmann E. B., Jr Standardization and control of meningococcal vaccines, group A and group C polysaccharides. J Biol Stand. 1977;5(3):197–215. doi: 10.1016/s0092-1157(77)80005-x. [DOI] [PubMed] [Google Scholar]
  38. Zitron I. M., Mosier D. E., Paul W. E. The role of surface IgD in the response to thymic-independent antigens. J Exp Med. 1977 Dec 1;146(6):1707–1718. doi: 10.1084/jem.146.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zollinger W. D., Mandrell R. E., Griffiss J. M., Altieri P., Berman S. Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man. J Clin Invest. 1979 May;63(5):836–848. doi: 10.1172/JCI109383. [DOI] [PMC free article] [PubMed] [Google Scholar]

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