title: Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule.
abstract: BACKGROUND
  Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by Haemophilus influenzae serotype b (Hib). This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination.
  
  
  RESULTS
  The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the hcsA and hcsB genes, involved in transport of capsular polysaccharide to the cell surface. Application of hcsA type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0-4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide.
  
  
  CONCLUSIONS
  Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide expression may have provided a selective advantage for type I strains resulting in the rapid elimination of type II from the Dutch Hib population after introduction of nationwide Hib vaccination. However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.
authors.affiliation: Laboratory for Infectious Diseases and Perinatal screening, National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, The Netherlands. LM.Schouls@rivm.nl
authors.forename: Leo
authors.initials: L
authors.lastname: Schouls
authors.forename: Han
authors.initials: H
authors.lastname: van der Heide
authors.forename: Sandra
authors.initials: S
authors.lastname: Witteveen
authors.forename: Bert
authors.initials: B
authors.lastname: Zomer
authors.forename: Arie
authors.initials: A
authors.lastname: van der Ende
authors.forename: Marina
authors.initials: M
authors.lastname: Burger
authors.forename: Corrie
authors.initials: C
authors.lastname: Schot
mesh_terms: D000368:Aged; D016667:Bacterial Capsules / Q000145:classification / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D005798:Genes, Bacterial; D014644:Genetic Variation; D006192:Haemophilus Infections / Q000382:microbiology* / Q000517:prevention & control; D018073:Haemophilus Vaccines / Q000008:administration & dosage; D019741:Haemophilus influenzae type b / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007223:Infant; D026901:Membrane Transport Proteins / Q000235:genetics; D009426:Netherlands / Q000453:epidemiology; D016133:Polymerase Chain Reaction; D011135:Polysaccharides, Bacterial / Q000235:genetics / Q000378:metabolism*; D012703:Serotyping; D014611:Vaccination
chemical_list: D018073:Haemophilus Vaccines; C511953:HcsA protein, Haemophilus influenzae; C511954:HcsB protein, Haemophilus influenzae; D026901:Membrane Transport Proteins; D011135:Polysaccharides, Bacterial