Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Dec;64(12):5187–5192. doi: 10.1128/iai.64.12.5187-5192.1996

Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media.

T F DeMaria 1, D M Murwin 1, E R Leake 1
PMCID: PMC174506  PMID: 8945564

Abstract

The role of nontypeable Haemophilus influenzae (NTHi) outer membrane protein (OMP) P6 in the pathogenesis of otitis media (OM) has not been defined. OMPs, fimbriae, pili, and lipooligosaccharide are several types of surface antigens of NTHi that are currently being evaluated as potential vaccine candidates. P6 is antigenically conserved among both nontypeable and type b H. influenzae strains and elicits bactericidal as well as protective antibodies; however, initial evaluation of a vaccine mixture of P6 combined with other NTHi OMPs failed to induce bactericidal antibody or protection in the chinchilla model of OM. We undertook an assessment of the ability of immunization with isolated P6 lipoprotein alone to confer protection. Chinchillas were immunized with P6 and challenged 10 days after the final immunization with either 3 x 10(3) CFU of NTHi delivered directly into the middle ear to induce OM or 5 x 10(8) CFU of NTHi delivered intranasally to establish nasopharyngeal colonization. All immunized animals responded with elevated serum titers of anti-P6 antibody, which also demonstrated bactericidal activity against homologous as well as a heterologous NTHi isolate. By 14 days post-transbullar challenge, the number of chinchillas with middle ear fluid and the incidence of NTHi culture-positive middle ear fluids were reduced 48 and 51%, respectively, in the P6-immunized chinchillas relative to the sham-immunized cohort. Nasopharyngeal colonization levels were comparable in the two cohorts. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM; however, parenteral immunization does not appear to affect the extent or duration of nasopharyngeal colonization by NTHi.

Full Text

The Full Text of this article is available as a PDF (238.5 KB).

Selected References

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

  1. Anderson P., Flesher A., Shaw S., Harding A. L., Smith D. H. Phenotypic and genetic variation in the susceptibility of Haemophilus influenzae type b to antibodies to somatic antigens. J Clin Invest. 1980 Apr;65(4):885–891. doi: 10.1172/JCI109741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bakaletz L. O., Tallan B. M., Andrzejewski W. J., DeMaria T. F., Lim D. J. Immunological responsiveness of chinchillas to outer membrane and isolated fimbrial proteins of nontypeable Haemophilus influenzae. Infect Immun. 1989 Oct;57(10):3226–3229. doi: 10.1128/iai.57.10.3226-3229.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barenkamp S. J. Protection by serum antibodies in experimental nontypable Haemophilus influenzae otitis media. Infect Immun. 1986 May;52(2):572–578. doi: 10.1128/iai.52.2.572-578.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clancy R. L., Cripps A. W. Specific protection against acute bronchitis associated with nontypeable Haemophilus influenzae. J Infect Dis. 1992 Jun;165 (Suppl 1):S194–S195. doi: 10.1093/infdis/165-supplement_1-s194. [DOI] [PubMed] [Google Scholar]
  5. DeMaria T. F. Animal models for nontypable Haemophilus influenzae otitis media. Pediatr Infect Dis J. 1989 Jan;8(1 Suppl):S40–S42. doi: 10.1097/00006454-198901001-00015. [DOI] [PubMed] [Google Scholar]
  6. DeMaria T. F., Briggs B. R., Lim D. J., Okazaki N. Experimental otitis media with effusion following middle ear inoculation of nonviable H influenzae. Ann Otol Rhinol Laryngol. 1984 Jan-Feb;93(1 Pt 1):52–56. doi: 10.1177/000348948409300113. [DOI] [PubMed] [Google Scholar]
  7. Deich R. A., Anilionis A., Fulginiti J., Metcalf B. J., Quataert S., Quinn-Dey T., Zlotnick G. W., Green B. A. Antigenic conservation of the 15,000-dalton outer membrane lipoprotein PCP of Haemophilus influenzae and biologic activity of anti-PCP antisera. Infect Immun. 1990 Oct;58(10):3388–3393. doi: 10.1128/iai.58.10.3388-3393.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Green B. A., Farley J. E., Quinn-Dey T., Deich R. A., Zlotnick G. W. The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene. Infect Immun. 1991 Sep;59(9):3191–3198. doi: 10.1128/iai.59.9.3191-3198.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Green B. A., Metcalf B. J., Quinn-Dey T., Kirkley D. H., Quataert S. A., Deich R. A. A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae. Infect Immun. 1990 Oct;58(10):3272–3278. doi: 10.1128/iai.58.10.3272-3278.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green B. A., Quinn-Dey T., Zlotnick G. W. Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b. Infect Immun. 1987 Dec;55(12):2878–2883. doi: 10.1128/iai.55.12.2878-2883.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green B. A., Vazquez M. E., Zlotnick G. W., Quigley-Reape G., Swarts J. D., Green I., Cowell J. L., Bluestone C. D., Doyle W. J. Evaluation of mixtures of purified Haemophilus influenzae outer membrane proteins in protection against challenge with nontypeable H. influenzae in the chinchilla otitis media model. Infect Immun. 1993 May;61(5):1950–1957. doi: 10.1128/iai.61.5.1950-1957.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Groeneveld K., van Alphen L., Voorter C., Eijk P. P., Jansen H. M., Zanen H. C. Antigenic drift of Haemophilus influenzae in patients with chronic obstructive pulmonary disease. Infect Immun. 1989 Oct;57(10):3038–3044. doi: 10.1128/iai.57.10.3038-3044.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hansen E. J., Hart D. A., McGehee J. L., Toews G. B. Immune enhancement of pulmonary clearance of nontypable Haemophilus influenzae. Infect Immun. 1988 Jan;56(1):182–190. doi: 10.1128/iai.56.1.182-190.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karasic R. B., Beste D. J., To S. C., Doyle W. J., Wood S. W., Carter M. J., To A. C., Tanpowpong K., Bluestone C. D., Brinton C. C., Jr Evaluation of pilus vaccines for prevention of experimental otitis media caused by nontypable Haemophilus influenzae. Pediatr Infect Dis J. 1989 Jan;8(1 Suppl):S62–S65. doi: 10.1097/00006454-198901001-00021. [DOI] [PubMed] [Google Scholar]
  15. Kyd J. M., Dunkley M. L., Cripps A. W. Enhanced respiratory clearance of nontypeable Haemophilus influenzae following mucosal immunization with P6 in a rat model. Infect Immun. 1995 Aug;63(8):2931–2940. doi: 10.1128/iai.63.8.2931-2940.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leake E. R., Holmes K., Lim D. J., DeMaria T. F. Peptidoglycan isolated from nontypeable Haemophilus influenzae induces experimental otitis media in the chinchilla. J Infect Dis. 1994 Dec;170(6):1532–1538. doi: 10.1093/infdis/170.6.1532. [DOI] [PubMed] [Google Scholar]
  17. Liu V. C., Smith A. L. Molecular mechanism of Haemophilus influenzae pathogenicity. Antibiot Chemother (1971) 1992;45:30–51. doi: 10.1159/000420999. [DOI] [PubMed] [Google Scholar]
  18. Munson R. S., Jr, Granoff D. M. Purification and partial characterization of outer membrane proteins P5 and P6 from Haemophilus influenzae type b. Infect Immun. 1985 Sep;49(3):544–549. doi: 10.1128/iai.49.3.544-549.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Murphy T. F., Apicella M. A. Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev Infect Dis. 1987 Jan-Feb;9(1):1–15. doi: 10.1093/clinids/9.1.1. [DOI] [PubMed] [Google Scholar]
  20. Murphy T. F., Bartos L. C., Campagnari A. A., Nelson M. B., Apicella M. A. Antigenic characterization of the P6 protein of nontypable Haemophilus influenzae. Infect Immun. 1986 Dec;54(3):774–779. doi: 10.1128/iai.54.3.774-779.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy T. F., Bartos L. C., Rice P. A., Nelson M. B., Dudas K. C., Apicella M. A. Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody. J Clin Invest. 1986 Oct;78(4):1020–1027. doi: 10.1172/JCI112656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murphy T. F., Campagnari A. A., Nelson M. B., Apicella M. A. Somatic antigens of Haemophilus influenzae as vaccine components. Pediatr Infect Dis J. 1989 Jan;8(1 Suppl):S66–S68. [PubMed] [Google Scholar]
  23. Murphy T. F., Kirkham C., Sikkema D. J. Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule. Infect Immun. 1992 May;60(5):2016–2022. doi: 10.1128/iai.60.5.2016-2022.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murphy T. F., Nelson M. B., Dudas K. C., Mylotte J. M., Apicella M. A. Identification of a specific epitope of Haemophilus influenzae on a 16,600-dalton outer membrane protein. J Infect Dis. 1985 Dec;152(6):1300–1307. doi: 10.1093/infdis/152.6.1300. [DOI] [PubMed] [Google Scholar]
  25. Rosenfeld R. M., Doyle W. J., Swarts J. D., Seroky J., Pinero B. P. Third-generation cephalosporins in the treatment of acute pneumococcal otitis media. An animal study. Arch Otolaryngol Head Neck Surg. 1992 Jan;118(1):49–52. doi: 10.1001/archotol.1992.01880010053015. [DOI] [PubMed] [Google Scholar]
  26. Sirakova T., Kolattukudy P. E., Murwin D., Billy J., Leake E., Lim D., DeMaria T., Bakaletz L. Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A. Infect Immun. 1994 May;62(5):2002–2020. doi: 10.1128/iai.62.5.2002-2020.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stjernquist-Desatnik A., Prellner K., Schalén C. Colonization by Haemophilus influenzae and group A streptococci in recurrent acute tonsillitis and in tonsillar hypertrophy. Acta Otolaryngol. 1990 Mar-Apr;109(3-4):314–319. doi: 10.3109/00016489009107448. [DOI] [PubMed] [Google Scholar]
  28. Suzuki K., Bakaletz L. O. Synergistic effect of adenovirus type 1 and nontypeable Haemophilus influenzae in a chinchilla model of experimental otitis media. Infect Immun. 1994 May;62(5):1710–1718. doi: 10.1128/iai.62.5.1710-1718.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  30. Wald E. R., Byers C., Guerra N., Casselbrant M., Beste D. Subacute sinusitis in children. J Pediatr. 1989 Jul;115(1):28–32. doi: 10.1016/s0022-3476(89)80324-5. [DOI] [PubMed] [Google Scholar]
  31. Yamanaka N., Faden H. Local antibody response to P6 of nontypable Haemophilus influenzae in otitis-prone and normal children. Acta Otolaryngol. 1993 Jul;113(4):524–529. doi: 10.3109/00016489309135857. [DOI] [PubMed] [Google Scholar]

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

RESOURCES