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Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule.

Schouls L, van der Heide H, Witteveen S, Zomer B, van der Ende A, Burger M, Schot C - BMC Microbiol. (2008)

Bottom Line: 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.NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences.However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.

View Article: PubMed Central - HTML - PubMed

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

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.

Conclusion: 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.

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Expression of the Hib polysaccharide capsule of type I and type II strains as measured by inhibition ELISA. The graph displays the OD values obtained in the ELISA after inhibition by the cell fraction and the culture supernatant obtained from type I and type II strains. Inhibition was performed with 3 different dilutions of the antigens. Each bar denotes the average of the values obtained with 4 different strains and the error bars indicate the standard error. Red bars, inhibition by type I cells; yellow, inhibition by type II cells; dark blue, inhibition by type I culture supernatant; light blue, inhibition by type II culture supernatant. Note that in this graph higher OD values represent lower polysaccharide expression.
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Figure 4: Expression of the Hib polysaccharide capsule of type I and type II strains as measured by inhibition ELISA. The graph displays the OD values obtained in the ELISA after inhibition by the cell fraction and the culture supernatant obtained from type I and type II strains. Inhibition was performed with 3 different dilutions of the antigens. Each bar denotes the average of the values obtained with 4 different strains and the error bars indicate the standard error. Red bars, inhibition by type I cells; yellow, inhibition by type II cells; dark blue, inhibition by type I culture supernatant; light blue, inhibition by type II culture supernatant. Note that in this graph higher OD values represent lower polysaccharide expression.

Mentions: In order to detect differences in the level of capsule polysaccharide expression by capsule locus type I and type II strains, polysaccharide production was assessed by an inhibition ELISA. Four type I and 4 type II strains were analyzed to determine the level of polysaccharide production. The results showed that absorption of serum with cell fractions of the type I cultures caused more inhibition than with cells of type II strains. Using a standard curve based on inhibition with serial dilutions of purified polysaccharide, we deduced that the type I cells contained approximately twice as much capsular polysaccharide as type II cells (Fig. 4). This result was reproducible (the same experiment was performed three times) and statistically significant (ANOVA analysis, p ≤ 0.002). The culture supernatants from type I and type II cultures contained significantly more polysaccharide than the cellular fractions. However, the amount of polysaccharide in the culture supernatant from type I strains did not differ significantly from that of type II strains. This indicates that the amount of shed polysaccharide in type I and type II strains is the same, but that the total amount of polysaccharide produced in type I strains is higher.


Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule.

Schouls L, van der Heide H, Witteveen S, Zomer B, van der Ende A, Burger M, Schot C - BMC Microbiol. (2008)

Expression of the Hib polysaccharide capsule of type I and type II strains as measured by inhibition ELISA. The graph displays the OD values obtained in the ELISA after inhibition by the cell fraction and the culture supernatant obtained from type I and type II strains. Inhibition was performed with 3 different dilutions of the antigens. Each bar denotes the average of the values obtained with 4 different strains and the error bars indicate the standard error. Red bars, inhibition by type I cells; yellow, inhibition by type II cells; dark blue, inhibition by type I culture supernatant; light blue, inhibition by type II culture supernatant. Note that in this graph higher OD values represent lower polysaccharide expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267795&req=5

Figure 4: Expression of the Hib polysaccharide capsule of type I and type II strains as measured by inhibition ELISA. The graph displays the OD values obtained in the ELISA after inhibition by the cell fraction and the culture supernatant obtained from type I and type II strains. Inhibition was performed with 3 different dilutions of the antigens. Each bar denotes the average of the values obtained with 4 different strains and the error bars indicate the standard error. Red bars, inhibition by type I cells; yellow, inhibition by type II cells; dark blue, inhibition by type I culture supernatant; light blue, inhibition by type II culture supernatant. Note that in this graph higher OD values represent lower polysaccharide expression.
Mentions: In order to detect differences in the level of capsule polysaccharide expression by capsule locus type I and type II strains, polysaccharide production was assessed by an inhibition ELISA. Four type I and 4 type II strains were analyzed to determine the level of polysaccharide production. The results showed that absorption of serum with cell fractions of the type I cultures caused more inhibition than with cells of type II strains. Using a standard curve based on inhibition with serial dilutions of purified polysaccharide, we deduced that the type I cells contained approximately twice as much capsular polysaccharide as type II cells (Fig. 4). This result was reproducible (the same experiment was performed three times) and statistically significant (ANOVA analysis, p ≤ 0.002). The culture supernatants from type I and type II cultures contained significantly more polysaccharide than the cellular fractions. However, the amount of polysaccharide in the culture supernatant from type I strains did not differ significantly from that of type II strains. This indicates that the amount of shed polysaccharide in type I and type II strains is the same, but that the total amount of polysaccharide produced in type I strains is higher.

Bottom Line: 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.NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences.However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.

View Article: PubMed Central - HTML - PubMed

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

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus