Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 Aug;59(8):2658–2663. doi: 10.1128/iai.59.8.2658-2663.1991

Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms.

M B Nelson 1, R S Munson Jr 1, M A Apicella 1, D J Sikkema 1, J P Molleston 1, T F Murphy 1
PMCID: PMC258070  PMID: 1713197

Abstract

Infections caused by Haemophilus influenzae are a major worldwide health problem. In particular, nontypeable strains of H. influenzae are a common cause of otitis media in infants and children. A vaccine to prevent these infections would result in the prevention of substantial morbidity and cost savings. A problem in identifying an appropriate vaccine antigen has been the enormous antigenic heterogeneity among nontypeable strains of H. influenzae. The present study was undertaken to characterize the conservation of the P6 outer membrane protein (approximately 16,000 daltons) among strains of H. influenzae. A total of 20 type b strains and 20 nontypeable strains of diverse geographic and clinical origins was studied. Three approaches were taken. (i) Antigenic determinants recognized by monoclonal and polyclonal antibodies were present on P6 in all 40 strains tested. The molecular weight of P6 was identical in all strains. (ii) Comparison of the DNA sequences of the P6 genes from three epidemiologically and serologically unrelated strains demonstrated 100% homology at the amino acid level and 97 to 99% homology at the nucleotide level. (iii) Restriction fragment length polymorphism analysis demonstrated that the P6 gene and flanking sequences were highly conserved among all strains. These three independent series of experiments indicated that the P6 protein is highly conserved among strains of H. influenzae. P6 should receive serious consideration for inclusion in a vaccine to prevent infections caused by nontypeable H. influenzae.

Full text

PDF
2658

Images in this article

2660Image
on p.2660
2661Image
on p.2661
2661Image
on p.2661

Selected References

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

  1. 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]
  2. Anderson P., Pichichero M. E., Insel R. A. Immunization of 2-month-old infants with protein-coupled oligosaccharides derived from the capsule of Haemophilus influenzae type b. J Pediatr. 1985 Sep;107(3):346–351. doi: 10.1016/s0022-3476(85)80504-7. [DOI] [PubMed] [Google Scholar]
  3. Anderson P., Pichichero M., Edwards K., Porch C. R., Insel R. Priming and induction of Haemophilus influenzae type b capsular antibodies in early infancy by Dpo20, an oligosaccharide-protein conjugate vaccine. J Pediatr. 1987 Nov;111(5):644–650. doi: 10.1016/s0022-3476(87)80237-8. [DOI] [PubMed] [Google Scholar]
  4. BERENBERG W., KEVY S. Acute epiglottitis in childhood; a serious emergency, readily recognized at the bedside. N Engl J Med. 1958 May 1;258(18):870–874. doi: 10.1056/NEJM195805012581802. [DOI] [PubMed] [Google Scholar]
  5. Berk S. L., Holtsclaw S. A., Wiener S. L., Smith J. K. Nontypeable Haemophilus influenzae in the elderly. Arch Intern Med. 1982 Mar;142(3):537–539. [PubMed] [Google Scholar]
  6. Bluestone C. D. Otitis media in children: to treat or not to treat? N Engl J Med. 1982 Jun 10;306(23):1399–1404. doi: 10.1056/NEJM198206103062305. [DOI] [PubMed] [Google Scholar]
  7. Campagnari A. A., Gupta M. R., Dudas K. C., Murphy T. F., Apicella M. A. Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae. Infect Immun. 1987 Apr;55(4):882–887. doi: 10.1128/iai.55.4.882-887.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dajani A. S., Asmar B. I., Thirumoorthi M. C. Systemic Haemophilus influenzae disease: an overview. J Pediatr. 1979 Mar;94(3):355–364. doi: 10.1016/s0022-3476(79)80571-5. [DOI] [PubMed] [Google Scholar]
  9. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Einhorn M. S., Weinberg G. A., Anderson E. L., Granoff P. D., Granoff D. M. Immunogenicity in infants of Haemophilus influenzae type B polysaccharide in a conjugate vaccine with Neisseria meningitidis outer-membrane protein. Lancet. 1986 Aug 9;2(8502):299–302. doi: 10.1016/s0140-6736(86)90001-2. [DOI] [PubMed] [Google Scholar]
  11. Eskola J., Peltola H., Takala A. K., Käyhty H., Hakulinen M., Karanko V., Kela E., Rekola P., Rönnberg P. R., Samuelson J. S. Efficacy of Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy. N Engl J Med. 1987 Sep 17;317(12):717–722. doi: 10.1056/NEJM198709173171201. [DOI] [PubMed] [Google Scholar]
  12. Faden H., Bernstein J., Brodsky L., Stanievich J., Krystofik D., Shuff C., Hong J. J., Ogra P. L. Otitis media in children. I. The systemic immune response to nontypable Hemophilus influenzae. J Infect Dis. 1989 Dec;160(6):999–1004. doi: 10.1093/infdis/160.6.999. [DOI] [PubMed] [Google Scholar]
  13. Ginsburg C. M., Howard J. B., Nelson J. D. Report of 65 cases of Haemophilus influenzae b pneumonia. Pediatrics. 1979 Sep;64(3):283–286. [PubMed] [Google Scholar]
  14. 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]
  15. Gyorkey F., Musher D., Gyorkey P., Goree A., Baughn R. Nontypable Haemophilus influenzae are unencapsulated both in vivo and in vitro. J Infect Dis. 1984 Apr;149(4):518–522. doi: 10.1093/infdis/149.4.518. [DOI] [PubMed] [Google Scholar]
  16. Haase E. M., Campagnari A. A., Sarwar J., Shero M., Wirth M., Cumming C. U., Murphy T. F. Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae. Infect Immun. 1991 Apr;59(4):1278–1284. doi: 10.1128/iai.59.4.1278-1284.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Howie V. M., Ploussard J. H., Sloyer J. The "otitis-prone" condition. Am J Dis Child. 1975 Jun;129(6):676–678. doi: 10.1001/archpedi.1975.02120430016006. [DOI] [PubMed] [Google Scholar]
  18. Lenoir A. A., Granoff P. D., Granoff D. M. Immunogenicity of Haemophilus influenzae type b polysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine in 2- to 6-month-old infants. Pediatrics. 1987 Aug;80(2):283–287. [PubMed] [Google Scholar]
  19. Marshall R., Teele D. W., Klein J. O. Unsuspected bacteremia due to Haemophilus influenzae: outcome in children not initially admitted to hospital. J Pediatr. 1979 Nov;95(5 Pt 1):690–695. doi: 10.1016/s0022-3476(79)80712-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Munson R., Jr, Grass S. Purification, cloning, and sequence of outer membrane protein P1 of Haemophilus influenzae type b. Infect Immun. 1988 Sep;56(9):2235–2242. doi: 10.1128/iai.56.9.2235-2242.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murphy T. F., Apicella M. A. Antigenic heterogeneity of outer membrane proteins of nontypable Haemophilus influenzae is a basis for a serotyping system. Infect Immun. 1985 Oct;50(1):15–21. doi: 10.1128/iai.50.1.15-21.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Murphy T. F., Bernstein J. M., Dryja D. M., Campagnari A. A., Apicella M. A. Outer membrane protein and lipooligosaccharide analysis of paired nasopharyngeal and middle ear isolates in otitis media due to nontypable Haemophilus influenzae: pathogenetic and epidemiological observations. J Infect Dis. 1987 Nov;156(5):723–731. doi: 10.1093/infdis/156.5.723. [DOI] [PubMed] [Google Scholar]
  27. Murphy T. F., Dudas K. C., Mylotte J. M., Apicella M. A. A subtyping system for nontypable Haemophilus influenzae based on outer-membrane proteins. J Infect Dis. 1983 May;147(5):838–846. doi: 10.1093/infdis/147.5.838. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Musher D. M., Kubitschek K. R., Crennan J., Baughn R. E. Pneumonia and acute febrile tracheobronchitis due to haemophilus influenzae. Ann Intern Med. 1983 Oct;99(4):444–450. doi: 10.7326/0003-4819-99-4-444. [DOI] [PubMed] [Google Scholar]
  30. Musser J. M., Barenkamp S. J., Granoff D. M., Selander R. K. Genetic relationships of serologically nontypable and serotype b strains of Haemophilus influenzae. Infect Immun. 1986 Apr;52(1):183–191. doi: 10.1128/iai.52.1.183-191.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Musser J. M., Granoff D. M., Pattison P. E., Selander R. K. A population genetic framework for the study of invasive diseases caused by serotype b strains of Haemophilus influenzae. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5078–5082. doi: 10.1073/pnas.82.15.5078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Musser J. M., Kroll J. S., Moxon E. R., Selander R. K. Evolutionary genetics of the encapsulated strains of Haemophilus influenzae. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7758–7762. doi: 10.1073/pnas.85.20.7758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nelson M. B., Apicella M. A., Murphy T. F., Vankeulen H., Spotila L. D., Rekosh D. Cloning and sequencing of Haemophilus influenzae outer membrane protein P6. Infect Immun. 1988 Jan;56(1):128–134. doi: 10.1128/iai.56.1.128-134.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Peltola H., Käyhty H., Virtanen M., Mäkelä P. H. Prevention of Hemophilus influenzae type b bacteremic infections with the capsular polysaccharide vaccine. N Engl J Med. 1984 Jun 14;310(24):1561–1566. doi: 10.1056/NEJM198406143102404. [DOI] [PubMed] [Google Scholar]
  36. Porras O., Caugant D. A., Gray B., Lagergård T., Levin B. R., Svanborg-Edén C. Difference in structure between type b and nontypable Haemophilus influenzae populations. Infect Immun. 1986 Jul;53(1):79–89. doi: 10.1128/iai.53.1.79-89.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Porras O., Caugant D. A., Lagergård T., Svanborg-Edén C. Application of multilocus enzyme gel electrophoresis to Haemophilus influenzae. Infect Immun. 1986 Jul;53(1):71–78. doi: 10.1128/iai.53.1.71-78.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stratton C. W., Hawley H. B., Horsman T. A., Tu K. K., Ackley A., Fernando N. K., Weinstein M. P. Hemophilus influenzae pneumonia in adults: report of five cases caused by ampicillin-resistant strains. Am Rev Respir Dis. 1980 Mar;121(3):595–598. doi: 10.1164/arrd.1980.121.3.595. [DOI] [PubMed] [Google Scholar]
  39. Teele D. W., Klein J. O., Rosner B., Bratton L., Fisch G. R., Mathieu O. R., Porter P. J., Starobin S. G., Tarlin L. D., Younes R. P. Middle ear disease and the practice of pediatrics. Burden during the first five years of life. JAMA. 1983 Feb 25;249(8):1026–1029. [PubMed] [Google Scholar]
  40. Tillotson J. R., Lerner A. M. Hemophilus influenzae bronchopneumonia in adults. Arch Intern Med. 1968 May;121(5):428–432. [PubMed] [Google Scholar]
  41. Verghese A., Berk S. L. Bacterial pneumonia in the elderly. Medicine (Baltimore) 1983 Sep;62(5):271–285. doi: 10.1097/00005792-198309000-00002. [DOI] [PubMed] [Google Scholar]
  42. Weinberg G. A., Einhorn M. S., Lenoir A. A., Granoff P. D., Granoff D. M. Immunologic priming to capsular polysaccharide in infants immunized with Haemophilus influenzae type b polysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine. J Pediatr. 1987 Jul;111(1):22–27. doi: 10.1016/s0022-3476(87)80336-0. [DOI] [PubMed] [Google Scholar]
  43. Weinberg G. A., Ghafoor A., Ishaq Z., Nomani N. K., Kabeer M., Anwar F., Burney M. I., Qureshi A. W., Musser J. M., Selander R. K. Clonal analysis of Hemophilus influenzae isolated from children from Pakistan with lower respiratory tract infections. J Infect Dis. 1989 Oct;160(4):634–643. doi: 10.1093/infdis/160.4.634. [DOI] [PubMed] [Google Scholar]
  44. Weinberg G. A., Granoff D. M. Polysaccharide-protein conjugate vaccines for the prevention of Haemophilus influenzae type b disease. J Pediatr. 1988 Oct;113(4):621–631. doi: 10.1016/s0022-3476(88)80369-x. [DOI] [PubMed] [Google Scholar]
  45. Weinberg G. A., Towler D. A., Munson R. S., Jr Lipoproteins of Haemophilus influenzae type b. J Bacteriol. 1988 Sep;170(9):4161–4164. doi: 10.1128/jb.170.9.4161-4164.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zlotnick G. W., Sanfilippo V. T., Mattler J. A., Kirkley D. H., Boykins R. A., Seid R. C., Jr Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae. J Biol Chem. 1988 Jul 15;263(20):9790–9794. [PubMed] [Google Scholar]

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

RESOURCES