Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jan;65(1):261–266. doi: 10.1128/iai.65.1.261-266.1997

Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression.

D C Reason 1, T C Wagner 1, A H Lucas 1
PMCID: PMC174585  PMID: 8975921

Abstract

To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.

Full Text

The Full Text of this article is available as a PDF (243.0 KB).

Selected References

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

  1. Atkinson M. J., Cowan M. J., Feeney A. J. New alleles of IGKV genes A2 and A18 suggest significant human IGKV locus polymorphism. Immunogenetics. 1996;44(2):115–120. [PubMed] [Google Scholar]
  2. Barbas C. F., 3rd, Björling E., Chiodi F., Dunlop N., Cababa D., Jones T. M., Zebedee S. L., Persson M. A., Nara P. L., Norrby E. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9339–9343. doi: 10.1073/pnas.89.19.9339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbas C. F., 3rd, Crowe J. E., Jr, Cababa D., Jones T. M., Zebedee S. L., Murphy B. R., Chanock R. M., Burton D. R. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10164–10168. doi: 10.1073/pnas.89.21.10164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barbas C. F., 3rd, Kang A. S., Lerner R. A., Benkovic S. J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7978–7982. doi: 10.1073/pnas.88.18.7978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burton D. R., Barbas C. F., 3rd Human antibodies to HIV-1 by recombinant DNA methods. Chem Immunol. 1993;56:112–126. [PubMed] [Google Scholar]
  6. Burton D. R., Barbas C. F., 3rd, Persson M. A., Koenig S., Chanock R. M., Lerner R. A. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10134–10137. doi: 10.1073/pnas.88.22.10134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caton A. J., Koprowski H. Influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6450–6454. doi: 10.1073/pnas.87.16.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Deng S. J., MacKenzie C. R., Sadowska J., Michniewicz J., Young N. M., Bundle D. R., Narang S. A. Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. J Biol Chem. 1994 Apr 1;269(13):9533–9538. [PubMed] [Google Scholar]
  10. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  11. Granoff D. M., Shackelford P. G., Holmes S. J., Lucas A. H. Variable region expression in the antibody responses of infants vaccinated with Haemophilus influenzae type b polysaccharide-protein conjugates. Description of a new lambda light chain-associated idiotype and the relation between idiotype expression, avidity, and vaccine formulation. The Collaborative Vaccine Study Group. J Clin Invest. 1993 Mar;91(3):788–796. doi: 10.1172/JCI116298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Insel R. A., Anderson P. W. Oligosaccharide-protein conjugate vaccines induce and prime for oligoclonal IgG antibody responses to the Haemophilus influenzae b capsular polysaccharide in human infants. J Exp Med. 1986 Feb 1;163(2):262–269. doi: 10.1084/jem.163.2.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Insel R. A., Kittelberger A., Anderson P. Isoelectric focusing of human antibody to the Haemophilus influenzae b capsular polysaccharide: restricted and identical spectrotypes in adults. J Immunol. 1985 Oct;135(4):2810–2816. [PubMed] [Google Scholar]
  14. Insel R. A., Varade W. S., Chu Y. W., Marin E., Fuleihan R., Geha R. S. Somatic mutation of human immunoglobulin V genes: bias, rate, and regulation. Ann N Y Acad Sci. 1995 Sep 29;764:158–169. doi: 10.1111/j.1749-6632.1995.tb55820.x. [DOI] [PubMed] [Google Scholar]
  15. Jeske D. J., Jarvis J., Milstein C., Capra J. D. Junctional diversity is essential to antibody activity. J Immunol. 1984 Sep;133(3):1090–1092. [PubMed] [Google Scholar]
  16. Lucas A. H., Azmi F. H., Mink C. M., Granoff D. M. Age-dependent V region expression in the human antibody response to the Haemophilus influenzae type b polysaccharide. J Immunol. 1993 Mar 1;150(5):2056–2061. [PubMed] [Google Scholar]
  17. Lucas A. H., Granoff D. M. Functional differences in idiotypically defined IgG1 anti-polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type B polysaccharide-protein conjugates. J Immunol. 1995 Apr 15;154(8):4195–4202. [PubMed] [Google Scholar]
  18. Lucas A. H., Langley R. J., Granoff D. M., Nahm M. H., Kitamura M. Y., Scott M. G. An idiotypic marker associated with a germ-line encoded kappa light chain variable region that predominates the vaccine-induced human antibody response to the Haemophilus influenzae b polysaccharide. J Clin Invest. 1991 Dec;88(6):1811–1818. doi: 10.1172/JCI115502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lucas A. H., Larrick J. W., Reason D. C. Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide. Infect Immun. 1994 Sep;62(9):3873–3880. doi: 10.1128/iai.62.9.3873-3880.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pinchuk G. V., Nottenburg C., Milner E. C. Predominant V-region gene configurations in the human antibody response to Haemophilus influenzae capsule polysaccharide. Scand J Immunol. 1995 Apr;41(4):324–330. doi: 10.1111/j.1365-3083.1995.tb03574.x. [DOI] [PubMed] [Google Scholar]
  21. Reason D. C., Lucas A. H. Content and dynamics of the human antibody variable region repertoire to the Haemophilus influenzae type b polysaccharide. Springer Semin Immunopathol. 1993;15(2-3):119–137. doi: 10.1007/BF00201096. [DOI] [PubMed] [Google Scholar]
  22. Scott M. G., Crimmins D. L., McCourt D. W., Chung G., Schäble K. F., Thiebe R., Quenzel E. M., Zachau H. G., Nahm M. H. Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous. J Immunol. 1991 Dec 1;147(11):4007–4013. [PubMed] [Google Scholar]
  23. Scott M. G., Crimmins D. L., McCourt D. W., Zocher I., Thiebe R., Zachau H. G., Nahm M. H. Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region. J Immunol. 1989 Dec 15;143(12):4110–4116. [PubMed] [Google Scholar]
  24. Williamson R. A., Burioni R., Sanna P. P., Partridge L. J., Barbas C. F., 3rd, Burton D. R. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4141–4145. doi: 10.1073/pnas.90.9.4141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zebedee S. L., Barbas C. F., 3rd, Hom Y. L., Caothien R. H., Graff R., DeGraw J., Pyati J., LaPolla R., Burton D. R., Lerner R. A. Human combinatorial antibody libraries to hepatitis B surface antigen. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3175–3179. doi: 10.1073/pnas.89.8.3175. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES