The importance of multiple Haemophilus influenzae...
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Children with Haemophilus influenzae type b (Hib) vaccine failure have long-term
bactericidal antibodies against virulent Hib strains with multiple capsular loci
Kelly Townsend-Payne1, Shamez N Ladhani2,3*, Helen Findlow1, Mary Slack45, Ray Borrow1,6
1 Vaccine Evaluation Unit, Public Health England, Clinical Sciences Building, Manchester
Royal Infirmary, Manchester, UK.
2 Immunisation Hepatitis and Blood Safety Department, Centre for Infectious Disease
Surveillance and Control, Public Health England, Colindale, London, UK.
3 Paediatric Infectious Diseases Research Group, St. George’s University of London, London,
UK
4 Institute of Hygiene and Microbiology, University of Wuerzburg
5 Department of Medicine, Griffith University, Queensland, Australia
6 Inflammation Sciences Research Group, University of Manchester, School of Translational
Medicine, Stopford Building, Manchester, UK.
*Corresponding author Dr Shamez Ladhani, Immunisation Hepatitis and Blood Safety
Department, Centre for Infectious Disease Surveillance and Control, Public Health England,
61 Colindale Avenue, London, UK. [email protected]
Keywords: Haemophilus influenzae type b, capsular operon, vaccine, failure
Running title: Serum bactericidal antibodies after Hib vaccine failure
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Abstract
Children who develop invasive Haemophilus influenzae serotype b (Hib) disease after
immunisation with a highly-effective conjugate vaccine are more likely to have been infected
with Hib strains possessing multiple copies of the capsulation locus. Using a recently-
validated serum bactericidal antibody (SBA) assay, we tested convalescent sera from 127 Hib
vaccine failure cases against clinical Hib strains expressing 1-5 copies of the capsulation
locus. SBA titres correlated weakly with anti-capsular IgG antibody concentrations and there
was no association between SBA geometric mean titres and number of capsulation locus
copies. After infection, children with Hib vaccine failure were equally protected against Hib
strains with 1-5 copies of the capsulation locus.
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Introduction
Encapsulated Haemophilus influenzae (Hi) have a polysaccharide capsule and can be
classified into six serotypes (a-f) based on antigenic differences. The genes for capsule
production reside within the capsulation (cap) locus, which consists of three functionally
defined regions [1]. Region 2 genes are involved in capsular biosynthesis and are unique to
each of the six capsule types [2]. Regions 1 and 3 are common to all six capsular types and
contain genes necessary for processing and transporting capsular material for surface
expression [2].
Of the encapsulated Hi, serotype b (Hib) is the most virulent and, prior to routine vaccination,
was responsible for the vast majority of invasive Hi infections, particularly meningitis in
young children [3]. Hib is more pathogenic than other Hi because its capsule consists of a
repeating polymer of ribosyl and ribitol phosphate (polyribosyl-ribitol-phosphate, PRP),
which enables the organism to effectively evade complement-mediated killing and avoid
splenic clearance. In the Hib chromosome, the capb locus is involved in PRP capsule
expression [4]. Most Hib isolates contain a partial duplication resulting in two complete
copies of regions 2 and 3, one complete copy of region 1 with a truncated copy of region 1
containing a 1.2-kb deletion within the bexA gene and IS1016, which is thought to stabilise
capsule production [1]. Most Hib isolates have two copies for detectable capsular expression
[5].
Hib conjugate vaccines contain PRP conjugated to a carrier protein and are very
immunogenic, resulting in high concentrations of antibodies against the PRP capsule [3],
which not only help prevent invasive disease at an individual level but, by reducing
nasopharyngeal carriage of Hib, also indirectly protect others through interrupting
transmission [3].
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The United Kingdom introduced the Hib conjugate vaccine into the national immunisation
programme in 1992 [3]. Unlike other countries, an accelerated infant Hib immunisation
schedule was implemented at 2-3-4 months of age without a booster in the second year of
life. Instead, a 12-month catch-up campaign targeting children up to 48 months of age was
implemented [3]. This programme was highly effective in reducing Hib disease across all
age-groups, through a combination of direct and indirect (herd) protection [6]. In vaccinated
children, invasive Hib disease was rare, with only 96 true vaccine failures (invasive Hib
disease after age-appropriate immunisation) identified during the first six years of the
programme (1992-98), equivalent to a vaccine failure rate of 2.2 cases per 100,000 vaccinees
[6]. Notably, however, at the time of infection, 54% of children with Hib vaccine failure had
anti-PRP IgG <0.15 g/mL and 92% had anti-PRP IgG <1.0 g/mL [11]. Since the Hib
conjugate vaccine only induced antibodies against the Hib PRP capsule, it follows that
children with low anti-PRP IgG at the time of infection with Hib would be at increased risk
of invasive disease, particularly if exposed to Hib strains with multiple capb copies that are
capable of producing large amounts of capsular polysaccharide [5].
Analysis of clinical Hib isolates showed that vaccinated children were significantly more
likely to be infected with a strain containing multiple capb copies than unvaccinated children
[7]. Moreover, the odds ratio for multiple-copy strains in children with Hib vaccine failure
increased with the number of capb copies in a dose-response manner when compared with
unvaccinated children [7].
Although Hib strains with three or more copies of the capb locus had previously been
isolated from patients with invasive disease [4], their association with vaccine failure was
novel. Isogenic strains with increasing capb copies have been shown to produce more capsule
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and be more resistant to complement-mediated, antibody-dependent bacteriolysis and
opsonisation by macrophages in vitro [4,5].
We recently followed-up UK children with Hib vaccine failure at a median of four years after
invasive Hib disease and found that more than half the children did not have sufficient anti-
PRP antibody to sustain long-term protection against invasive Hib disease [8]. We were,
therefore, concerned that children with Hib vaccine failure might, in the long-term, become
susceptible to further episodes of invasive Hib disease with more virulent strains possessing
multiple capb copies. In this study, we tested sera from children with Hib vaccine failure
against invasive clinical isolates expressing 1 to 5 capb copies using a recently-validated Hib
serum bactericidal antibody (SBA) assay [9].
Materials and Methods
Public Health England (PHE) conducts enhanced national surveillance of invasive Hi disease
in England and Wales. As part of the surveillance, the PHE Haemophilus Reference Unit
provides a free national service for confirmation and serotyping of invasive sterile-site H.
influenzae isolates. The number of capb copies in clinical isolates from children aged <60
months, irrespective of their immunisation status, was determined in a previous study [7].
One strain each with 1, 2, 3, 4 and 5 capb copies was randomly selected for the current study.
Hib vaccine failure was defined as invasive Hib disease occurring any time after three
vaccine doses in the first year of life, or >1 week after two doses in the first year, or >2 weeks
after one dose at >12 months of age [6]. Sera for this study were obtained from 164 children
with Hib vaccine failure as described previously [8]. Briefly, children with Hib vaccine
failure diagnosed between October 1992 and December 2005 were identified through
national surveillance and followed-up at a median of 4.1 (interquartile range, 3.5–9.7) years
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after their infection. In keeping with national guidelines, children with Hib vaccine failure are
recommended to receive an additional dose of Hib conjugate vaccine after recovering from
their infection. Following permission from their general practitioner and written consent from
the parents, the children had one blood sample taken. The sera were tested against Hib strains
containing 1, 2, 3, 4 or 5 capb copies using a recently-validated Hib SBA assay [9], except
for a change from the T50 to T60 time point. In brief, serum samples were tested with a
starting dilution of 1:8. Two-fold serum serial dilutions were made in 15 μl of Bactericidal
buffer (BB) supplemented with 2% Fildes enrichment. Each bacterial strain grown to log
phase, was diluted to yield 2.4- 3.4 x 108 CFU/mL and 10µL was added to each well. Baby
rabbit complement was added to each well except the CFU and serum control wells, to which
heat-inactivated baby rabbit complement was added at a concentration of 33.3%. An
additional 15 μl of the dilution buffer was added to each well to bring the total volume of the
reaction to 60μL. After an incubation of 60 min (T60) at 37°C, 10μL from each well was
plated onto chocolate agar plates. After 16 h of incubation at 37°C in 5% CO2, the colony
growth on each chocolate agar plate was enumerated using Sorcerer Colony Counter
(Perceptive Instruments). The SBA titre for each unknown serum sample was expressed as
the reciprocal serum dilution yielding ≥50% killing compared to the number of target cells
present in the CFU control well.
SBA geometric mean titres (GMTs) with 95% confidence intervals (CIs) and percentage of
children having SBA titres of ≥8 were calculated for samples assayed against each strain. The
SBA titres for each strain were correlated against known anti-PRP IgG concentrations
measured by a Hib microsphere immunoassay [10].
Linear regression was used to assess correlation between logged anti-PRP IgG concentrations
(the dependent variable) and number of strains protected (Hib SBA ≥8 against the test
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strain), after adjusting for the number of capb copies (independent variables). Logistic
regression was used to assess the association between the proportion of children with anti-
PRP IgG ≥0.15 g/mL (the putative short-term protective threshold against invasive Hib
disease) and with the number of strains protected, after adjusting for the number of capb
copies. Logistic regression was repeated for the proportion of children with anti-PRP IgG
≥1.0 g/mL, the putative long-term protective threshold against invasive Hib disease.
Ethics Approval
Ethical agreement was obtained from the Thames Valley Multi-Centre Research Ethics
Committee (reference: 05/MRE12/50).
Results
Sufficient sera were available for 127 of 164 children with Hib vaccine failure. Anti-PRP IgG
concentrations between the 127 children with sufficient sera and the 37 children with
insufficient sera were not significantly different (data not shown). Of the 127 children with
sufficient serum, the overall anti-PRP IgG GMC was 0.98 (95% CI, 0.65-1.48), with 80% and
46% having concentrations ≥0.15 g/mL and ≥1.0 g/mL, respectively. Hib SBA titres
correlated weakly with previously determined anti-PRP IgG for individual Hib strains
containing 1, 2, 3, 4, or 5 capb copies (Table 1). There was no correlation between number of
capb copies and proportions having Hib SBA ≥8 or anti-PRP IgG ≥0.15 g/mL. A large
proportion (43-54%) of children had both anti-PRP IgG ≥0.15 g/mL and SBA ≥8 for each
of the tested strains with different capb copies. A substantial proportion (26-38%) had
protective IgG concentrations (≥0.15 g/mL) with low SBA titres (<8), while a smaller
proportion (3-8%) had low IgG concentrations (<0.15 g/mL) but high SBA titres (Table 1).
Irrespective of capb copy number, however, there was a significant correlation between the
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number of strains protected against (SBA ≥8) and logged anti-PRP IgG (P<0.0001). There
was also a significant association with proportions having anti-PRP IgG ≥0.15 g/mL
(P=0.006) and ≥1.0 g/mL (P<0.0001), respectively (Table 2).
Discussion
Most strains isolated from patients with invasive Hib disease generally posses a duplication
of the cap b locus, however further amplification has been detected (three, four, five, six
copies) and has thought to play a role in in Hib vaccine failure (7). After invasive disease,
most children with Hib vaccine failure had high anti-PRP IgG concentrations and, of those
who didn’t, the majority responded to a further dose of Hib conjugate vaccine [11]. However,
when we followed-up children with Hib vaccine failure at a median of 4 years after their
infection, we found that 16% and 57% had anti-PRP IgG <0.15 g/mL and <1.0 g/mL,
respectively [8]. We were, therefore, concerned that such children might, once again, become
susceptible to invasive Hib disease, especially if they were exposed to more virulent Hib
strains possessing more than two copies of the cap b locus. This, however, appears not to be
the case since we found no significant correlation between capb copy numbers and Hib SBA
titres in this study.
Previous in vitro studies showed that isogenic Hib strains became more resistant to antibody-
dependent, classical complement pathway-directed bacteriolysis and opsonisation with
increasing capb copy numbers [5]. An important difference with our study is that we assessed
SBA activity against clinical isolates with different capb copy numbers and not against
isogenic strains. It has been proposed that strains with an amplified capb locus produce more
capsular polysaccharide and are, therefore, more capable of overcoming host defense and
invading the bloodstream of immunised children, especially those with suboptimal anti-PRP
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antibody concentrations (7). In this study, we had hypothesised that Hib strains containing
more capb copy numbers would be more resistant to complement mediated killing in the
SBA assay using sera from children with Hib vaccine failure.
Since the SBA assay measures functional activity against the whole organism and not only
against the capsular polysaccharide, it is likely that children with Hib vaccine failure develop
a range of functional antibodies against other Hib subcapsular antigens, such as P1, P2, P4,
P6 and protein D [12]. This might explain why there was no clear correlation between SBA
titres and capb copy numbers, but demonstrates the usefulness of the SBA assay to
complement anti-capsular IgG concentrations when assessing functional protection against
encapsulated bacteria.
Rare meningococcal serogroup C (MenC) strains that resist killing by bactericidal antibodies
induced by a MenC conjugate vaccine have also been identified [13]. These strains have an
insertion sequence, IS1301, which amplifies transcription levels of surrounding genes and the
amount of capsule produced, resulting in down-regulation of the alternative complement
pathway [13]. Unlike Hib, however, such strains were not identified among MenC vaccine
failure cases in the UK and were not more common among carriage isolates compared to
disease isolates [14]. While increased capsule production may provide a survival advantage
for the bacteria, such strains do not appear to be widespread among invasive cases, perhaps
because this may be detrimental at other stages of their lifecycle or may be more effectively
cleared by other host immune mechanisms [13].
A limitation of our study is that we do not know the capsulation status of the infecting Hib
strains in individual children with Hib vaccine failure who provided the serum samples,
which would have been interesting to correlate with anti-PRP IgG and SBA titres. Hib strains
with multiple copies that produce more capsule could drive a stronger inflammatory response
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leading to higher post-infection anti-PRP antibodies and more robust long-term protective
response.
Another limitation is that we were unable to measure the amount of capsular polysaccharide
produced by the Hib strains with different capb copy numbers and, therefore, cannot be
certain that actually did produce different amounts of capsule. Moreover, we did not have
isogenic strains, which would have allowed more objective assessment of responses against
capb copy numbers. However, by using clinical isolates, we were able to assess protection
against circulating strains that potential cases are likely to be exposed to. Finally, it would be
interesting to assess functional activity against Hib strains with increasing capb copy
numbers using sera from immunised children who did not develop invasive disease, since
such children would only possess vaccine-induced anti-PRP IgG [9].
In conclusion, children who develop invasive Hib disease after immunisation with a highly
effective conjugate vaccine develop protective bactericidal antibodies against other surface
antigens in addition to the PRP capsule. In a recently validated Hib SBA assay using sera
from children with Hib vaccine failure several years after their infection, we found no
association between bactericidal activity and clinical Hib isolates with multiple capb copies.
We, however, did find that children with higher anti-PRP IgG concentrations were more
likely to be protected against more Hib strains of different genetic lineages, irrespective of
capb copy number. This finding highlights the importance of having and sustaining high
antibody concentrations after vaccination to ensure long-term individual protection and
population control of vaccine-preventable bacterial infections [15].
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Acknowledgements: The authors would like to thank the General Practitioners,
paediatricians and families of all children with Hib vaccine failure who participated in this
study. Dr Shamez Ladhani was awarded a competitive two-year European Society for
Paediatric Infectious Diseases (ESPID) to study Hib vaccine failure in children.
Funding: none
Conflicts of Interest: none
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Table 1. Serum bactericidal activity against Haemophilus influenzae type b (Hib) strains with increasing numbers of the capb locus
using sera from 127 children with Hib vaccine failure at a median of four years after infection.
Number of copies
of capb locus
Hib SBA GMT
(95% CI)
SBA ≥8,
N (%)
IgG ≥0.15 g/mL
and SBA <8
Both IgG ≥0.15
g/mL and SBA ≥8
SBA≥8 and IgG
<0.15 g/mL
SBA<8 and IgG
<0.15 g/mL
Correlation between anti-
PRP IgG and Hib SBA *
1 46 (29-79) 79 (62%) 33 (26%) 69 (54%) 10 (8%) 15 (12%) 0.535
2 24 (15-38) 62 (49%) 47 (37%) 55 (43%) 7 (6%) 18 (14%) 0.457
3 43 (27-69) 74 (58%) 37 (29%) 65 (51%) 9 (7%) 16 (13%) 0.509
4 55 (33-90) 77 (61%) 35 (28%) 67 (53%) 10 (8%) 15 (12%) 0.549
5 20 (13-31) 58 (46%) 48 (38%) 54 (43%) 4 (3%) 21 (17%) 0.595
Hib, Haemophilus influenzae type b; SBA, serum bactericidal antibody; GMT, geometric mean titre; IgG, Immunoglobulin G; PRP, polyribosyl-
ribitol-phosphate (the Hib polysaccharide capsule)
* The Hib SBA titres and anti PRP IgG concentrations were correlated using Pearson`s correlation coefficients (r value)
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Table 2. Protection against Haemophilus influenzae type b (Hib) clinical isolates in a recently-developed serum bactericidal antibody
(SBA) assay using sera from 127 children with Hib vaccine failure at a median of four years after infection.
Number of strains
protected against *
Number
of cases
Anti-PRP IgG GMC
(95% CI)
Anti-PRP IgG
≥0.15g/mL, N (%)
Anti-PRP IgG ≥1.0
g/mL, N (%)
None 46 0.35 (0.25-0.51) 32 (90%) 9 (20%)
1-2 strains 7 0.46 (0.09-2.37) 5 (71%) 2 (29%)
3-4 strains 22 0.99 (0.41-2.38) 16 (73%) 9 (41%)
All 5 strains 52 4.33 (2.47-7.58) 49 (94%) 39 (75%)
TOTAL 127 1.25 (0.86-1.80) 102 (80%) 59 (46%)
* Defined as Hib serum bactericidal antibody (SBA) ≥8 against the test strains. The five strains include clinical Hib isolates with 1-5 copies of
the capb locus
Hib, Haemophilus influenzae type b; PRP, polyribosyl-ribitol-phosphate (the Hib polysaccharide capsule) GMC, geometric mean
concentration; IgG, Immunoglobulin G
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