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. 1996 Sep;64(9):3576–3583. doi: 10.1128/iai.64.9.3576-3583.1996

Variation in repeat number within the alpha C protein of group B streptococci alters antigenicity and protective epitopes.

C Gravekamp 1, D S Horensky 1, J L Michel 1, L C Madoff 1
PMCID: PMC174266  PMID: 8751902

Abstract

Variable expression of repeating units of the protective alpha C proteins among clinical isolates of group B streptococci (GBS) may have implications for vaccine development. In this study, alpha C protein genes containing various numbers of repeats (1,2,9, and 16) were cloned in a T7 overexpression vector in Escherichia coli. Expression was induced by isopropyl-beta-D-thiogalactopyranoside, and proteins were purified by anion-exchange, gel filtration, or affinity chromatography or by isoelectric focusing. Rabbits were immunized with purified 1-,2-,9-, or 16-repeat proteins. All proteins appeared to be highly immunogenic. Enzyme-linked immunosorbent assay inhibition with 9-repeat protein as the coating antigen and 9-repeat-antigen-elicited antiserum showed that a 200-fold-higher concentration of 1-repeat antigen than of 9- or 16-repeat antigen was required for 50% inhibition of antibody-antigen binding. The concentration of 2-repeat antigen required for 50% inhibition was intermediate relative to the concentrations of 1- and 9-repeat antigens. These results suggested that antibodies to 9-repeat antigen recognized predominantly a conformational epitope(s) contained in proteins with higher numbers of repeats (9 or 16) but lost considerable binding affinities for an epitope(s) contained in alpha C proteins with fewer repeats (1 or 2). Similar results were obtained with antiserum to 16-repeat antigen. However, antibodies to 1- and 2-repeat antigens recognized 1-,2-,9-,and 16-repeat antigens with equal binding affinities. This finding suggested that 1- and 2-repeat-elicited antibodies recognized an epitope(s) on individual repeats. Loss of repeating units from the alpha C proteins may result in decreased protection because the loss of epitopes (including conformational epitopes) gives the microorganisms the opportunity to escape host antibodies. If 1- and 2-repeat-elicited antibodies bind all alpha C proteins with equal affinity, regardless of their repeat number, they may prevent GBS strains with fewer repeats from escaping host immunity. Protection data obtained with antisera to the proteins with different repeat numbers support this hypothesis: mouse pups challenged with GBS strain A909 were better protected when immunized with 1- or 2-repeat-elicited antiserum (76 and 75%, respectively) than when immunized with 9- or 16-repeat-elicited antiserum (41 and 48%, respectively).

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Selected References

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  1. Anderson B. E., McDonald G. A., Jones D. C., Regnery R. L. A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences. Infect Immun. 1990 Sep;58(9):2760–2769. doi: 10.1128/iai.58.9.2760-2769.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bevanger L., Maeland J. A. Complete and incomplete Ibc protein fraction in group B streptococci. Acta Pathol Microbiol Scand B. 1979 Feb;87B(1):51–54. doi: 10.1111/j.1699-0463.1979.tb02402.x. [DOI] [PubMed] [Google Scholar]
  3. Broekhuijsen M. P., van Rijn J. M., Blom A. J., Pouwels P. H., Enger-Valk B. E., Brown F., Francis M. J. Fusion proteins with multiple copies of the major antigenic determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals. J Gen Virol. 1987 Dec;68(Pt 12):3137–3143. doi: 10.1099/0022-1317-68-12-3137. [DOI] [PubMed] [Google Scholar]
  4. Campbell J. L., Richardson C. C., Studier F. W. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc Natl Acad Sci U S A. 1978 May;75(5):2276–2280. doi: 10.1073/pnas.75.5.2276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crain M. J., Waltman W. D., 2nd, Turner J. S., Yother J., Talkington D. F., McDaniel L. S., Gray B. M., Briles D. E. Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae. Infect Immun. 1990 Oct;58(10):3293–3299. doi: 10.1128/iai.58.10.3293-3299.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gilmore R. D., Jr Comparison of the rompA gene repeat regions of Rickettsiae reveals species-specific arrangements of individual repeating units. Gene. 1993 Mar 15;125(1):97–102. doi: 10.1016/0378-1119(93)90752-o. [DOI] [PubMed] [Google Scholar]
  7. Hollingshead S. K., Fischetti V. A., Scott J. R. Size variation in group A streptococcal M protein is generated by homologous recombination between intragenic repeats. Mol Gen Genet. 1987 May;207(2-3):196–203. doi: 10.1007/BF00331578. [DOI] [PubMed] [Google Scholar]
  8. Ibañez C. F., Affranchino J. L., Macina R. A., Reyes M. B., Leguizamon S., Camargo M. E., Aslund L., Pettersson U., Frasch A. C. Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs. Mol Biochem Parasitol. 1988 Jul;30(1):27–33. doi: 10.1016/0166-6851(88)90129-6. [DOI] [PubMed] [Google Scholar]
  9. Johnson D. R., Ferrieri P. Group B streptococcal Ibc protein antigen: distribution of two determinants in wild-type strains of common serotypes. J Clin Microbiol. 1984 Apr;19(4):506–510. doi: 10.1128/jcm.19.4.506-510.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones K. F., Hollingshead S. K., Scott J. R., Fischetti V. A. Spontaneous M6 protein size mutants of group A streptococci display variation in antigenic and opsonogenic epitopes. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8271–8275. doi: 10.1073/pnas.85.21.8271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kemp D. J., Coppel R. L., Stahl H. D., Bianco A. E., Corcoran L. M., McIntyre P., Langford C. J., Favaloro J. M., Crewther P. E., Brown G. V. The Wellcome Trust lecture. Genes for antigens of Plasmodium falciparum. Parasitology. 1986;92 (Suppl):S83–108. doi: 10.1017/s0031182000085711. [DOI] [PubMed] [Google Scholar]
  12. Khan C. M., Villarreal-Ramos B., Pierce R. J., Demarco de Hormaeche R., McNeill H., Ali T., Chatfield S., Capron A., Dougan G., Hormaeche C. E. Construction, expression, and immunogenicity of multiple tandem copies of the Schistosoma mansoni peptide 115-131 of the P28 glutathione S-transferase expressed as C-terminal fusions to tetanus toxin fragment C in a live aro-attenuated vaccine strain of Salmonella. J Immunol. 1994 Dec 15;153(12):5634–5642. [PubMed] [Google Scholar]
  13. Klein B. S., Hogan L. H., Jones J. M. Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis. J Clin Invest. 1993 Jul;92(1):330–337. doi: 10.1172/JCI116571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lancefield R. C., McCarty M., Everly W. N. Multiple mouse-protective antibodies directed against group B streptococci. Special reference to antibodies effective against protein antigens. J Exp Med. 1975 Jul 1;142(1):165–179. doi: 10.1084/jem.142.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levy N. J., Nicholson-Weller A., Baker C. J., Kasper D. L. Potentiation of virulence by group B streptococcal polysaccharides. J Infect Dis. 1984 Jun;149(6):851–860. doi: 10.1093/infdis/149.6.851. [DOI] [PubMed] [Google Scholar]
  16. Madoff L. C., Hori S., Michel J. L., Baker C. J., Kasper D. L. Phenotypic diversity in the alpha C protein of group B streptococci. Infect Immun. 1991 Aug;59(8):2638–2644. doi: 10.1128/iai.59.8.2638-2644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Madoff L. C., Michel J. L., Gong E. W., Kling D. E., Kasper D. L. Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4131–4136. doi: 10.1073/pnas.93.9.4131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Madoff L. C., Michel J. L., Gong E. W., Rodewald A. K., Kasper D. L. Protection of neonatal mice from group B streptococcal infection by maternal immunization with beta C protein. Infect Immun. 1992 Dec;60(12):4989–4994. doi: 10.1128/iai.60.12.4989-4994.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Madoff L. C., Michel J. L., Kasper D. L. A monoclonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci. Infect Immun. 1991 Jan;59(1):204–210. doi: 10.1128/iai.59.1.204-210.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Michel J. L., Madoff L. C., Olson K., Kling D. E., Kasper D. L., Ausubel F. M. Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10060–10064. doi: 10.1073/pnas.89.21.10060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nickoloff J. A. Sepharose spin column chromatography. A fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation. Mol Biotechnol. 1994 Feb;1(1):105–108. doi: 10.1007/BF02821513. [DOI] [PubMed] [Google Scholar]
  22. Rakonjac J. V., Robbins J. C., Fischetti V. A. DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain. Infect Immun. 1995 Feb;63(2):622–631. doi: 10.1128/iai.63.2.622-631.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Relman D. A., Domenighini M., Tuomanen E., Rappuoli R., Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2637–2641. doi: 10.1073/pnas.86.8.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rodewald A. K., Onderdonk A. B., Warren H. B., Kasper D. L. Neonatal mouse model of group B streptococcal infection. J Infect Dis. 1992 Sep;166(3):635–639. doi: 10.1093/infdis/166.3.635. [DOI] [PubMed] [Google Scholar]
  25. Roditi I., Carrington M., Turner M. Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature. 1987 Jan 15;325(6101):272–274. doi: 10.1038/325272a0. [DOI] [PubMed] [Google Scholar]
  26. Schneider A., Hemphill A., Wyler T., Seebeck T. Large microtubule-associated protein of T. brucei has tandemly repeated, near-identical sequences. Science. 1988 Jul 22;241(4864):459–462. doi: 10.1126/science.3393912. [DOI] [PubMed] [Google Scholar]
  27. Signäs C., Raucci G., Jönsson K., Lindgren P. E., Anantharamaiah G. M., Hök M., Lindberg M. Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc Natl Acad Sci U S A. 1989 Jan;86(2):699–703. doi: 10.1073/pnas.86.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  29. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  30. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Timoney J. F., Walker J., Zhou M., Ding J. Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus. Infect Immun. 1995 Apr;63(4):1440–1445. doi: 10.1128/iai.63.4.1440-1445.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wallis A. E., McMaster W. R. Identification of Leishmania genes encoding proteins containing tandemly repeating peptides. J Exp Med. 1987 Dec 1;166(6):1814–1824. doi: 10.1084/jem.166.6.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Watson H. L., Blalock D. K., Cassell G. H. Variable antigens of Ureaplasma urealyticum containing both serovar-specific and serovar-cross-reactive epitopes. Infect Immun. 1990 Nov;58(11):3679–3688. doi: 10.1128/iai.58.11.3679-3688.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wessels M. R., Muñoz A., Kasper D. L. A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9170–9174. doi: 10.1073/pnas.84.24.9170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yother J., Briles D. E. Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis. J Bacteriol. 1992 Jan;174(2):601–609. doi: 10.1128/jb.174.2.601-609.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zheng X., Teng L. J., Watson H. L., Glass J. I., Blanchard A., Cassell G. H. Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigen size variation. Infect Immun. 1995 Mar;63(3):891–898. doi: 10.1128/iai.63.3.891-898.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zheng X., Watson H. L., Waites K. B., Cassell G. H. Serotype diversity and antigen variation among invasive isolates of Ureaplasma urealyticum from neonates. Infect Immun. 1992 Aug;60(8):3472–3474. doi: 10.1128/iai.60.8.3472-3474.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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