Objectives:

H. influenzae type b (Hib) strains isolated from invasive disease generally possess a duplication of the capsule (cap b) locus. Amplification up to five copies has been reported and has been proposed to be a mechanism to evade host defence. To verify if amplification of cap b locus is involved in vaccine failure we determined the number of copies of the cap b locus in Hib isolates from true vaccine failures (TVFs) and from age-matched controls.

Methods:

A total of 189 invasive Hib strains isolated from infants or children in the UK and the Republic of Ireland were tested. Of these, 95 were from patients with TVFs and 94 from age-matched controls. The copy number of cap b locus was determined by Southern blot analysis, using as a probe the 480 bp amplicon (capsule type b-specific) obtained by PCR of the Hib strain Eagan. As the KpnI and SmaI sites flank the cap b locus, the copy number can be estimated by the size of the restriction fragment obtained following digestion of the chromosome with these enzymes. The DNA fragment for a two-copy strain was expected to be approximately 45 kbp; strains with three or more copies of the locus featured fragments of increased size (63, 81 and 99 kbp).

Results:

Most isolates both in the TVFs and the control group exhibited hybridisation signals at the expected position for a two-copy arrangement of cap b locus. However, besides the 45 kbp band, several isolates containing the two-copy arrangement showed strong hybridisation signals at molecular weights higher than 50 kbp, indicating the presence of multiple copies (three, four and five repeats) of the locus. A significantly greater proportion of isolates from patients with TVFs contained multiple copies compared with strains from controls. In fact, 24/95 strains harboured three or more copies of the locus in the TVF group, vs. 11/94 among controls (P = 0.016). Interestingly, the presence of multiple copies was significantly associated with a greater proportion of clinical presentations other than meningitis in children belonging to both the TVFs and control groups.

Conclusions:

Our results show the number of multiple copy strains found among TVFs was significantly higher than in the control group. Although cases of invasive Hib disease in vaccinated children have been generally related to clinical or immunological conditions of the host or the use of less immunogenic vaccines, these data suggest that amplification of cap b locus may also be involved.