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. shamez.ladhani@phe.gov.uk

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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