Vaccine 24 (2006) 5335–5340

Influence of genetic and environmental factors on theimmunogenicity of Hib vaccine in Gambian twins

Y.C. Lee a, M.J. Newport b,c, T. Goetghebuer b,d, C.A. Siegrist e,H.A. Weiss f, A.J. Pollard a, A. Marchant b,g,∗,

the MRC Twin Study Group1

a Department of Paediatrics, University of Oxford, United Kingdomb Medical Research Council Laboratories, The Gambia

c Department of Medicine, Brighton and Sussex Medical School, Brighton, United Kingdomd Department of Pediatrics, Hopital Saint-Pierre, Brussels, Belgium

e WHO Collaborative Centre for Neonatal Vaccinology, University of Geneva, Switzerlandf MRC Tropical Epidemiology Unit, London School of Hygiene and Tropical Medicine, United Kingdom

g Institute for Medical Immunology, Universite Libre de Bruxelles, Belgium

Received 30 January 2006; received in revised form 4 April 2006; accepted 9 April 2006Available online 2 May 2006

Abstract

The differences in incidence rates of Haemophilus influenzae type b disease and the variation in Hib conjugate vaccine efficacy achievedamong different ethnic groups suggest genetic influences on the immune response to Hib vaccine. The serum anti-PRP antibody concentrationof 43 monozygotic (MZ) and 147 dizygotic (DZ) twin pairs in the Gambia was measured using a standardised Hib ELISA. Intrapair correlationsfor MZ and DZ twin pairs were compared and heritability in antibody responses to Hib conjugate vaccine was estimated to be 51% (95% CI:32–66%), indicating a significant genetic contribution in the response. We conclude that genetic factors may be involved in the variation inimmune response to Hib vaccine observed in different populations and may contribute to cases of vaccine failure.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Haemophilus influenzae vaccine; Antibody; Genetic predisposition

∗ Corresponding author at: Institute for Medical Immunology, Rue Adri-enne Bolland 8, 6041 Gosselies, Belgium. Tel.: +32 2 650 9588;fax: +32 2 650 9563.

E-mail address: armarcha@ulb.ac.be (A. Marchant).1 A. Allen, W. Banya, D. Jackson Sillah, K.P.W.J. McAdam, M. Mendy, M.

Ota, J. Vekemans, H. Whittle (The Medical Research Council Laboratories,The Gambia); K. Jobe (The Gambian Expanded Programme on Immunisa-tion, Department of State for Health, Banjul, The Gambia); S. Bennett (TheMRC Tropical Epidemiology Unit, London School of Hygiene and Trop-ical Medicine, UK); P. Aaby (The Danish Epidemiology Science Centre,Statens Serum Institut, Copenhagen, Denmark); J.C. Stockton (Department

1. Introduction

Haemophilus influenzae type b (Hib) was once the lead-ing global cause of bacterial meningitis, epiglottitis, septicarthritis, pneumonia, pericarditis, and facial cellulitis in chil-dren younger than 5 years of age. The introduction of the Hibconjugate vaccine, the first polysaccharide-protein conjugatevaccine, into routine childhood immunization regimens hassubstantially changed the epidemiology of invasive Hib dis-ease. There are a range of vaccines types, strategies of intro-duction, and immunization schedules in different countries.Nevertheless, all participating countries have experienced a

dramatic decline in the incidence of invasive Hib disease of

of Medicine, University of Cambridge, UK); G. Cadau, P.-H. Lambert, S.Schlegel-Hauter, P. Valenti (The WHO Collaborative Centre for NeonatalVaccinology, University of Geneva, Switzerland).

more than 90% since the introduction of Hib conjugate vac-cine [1].

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2006.04.021

5336 Y.C. Lee et al. / Vaccine 24 (2006) 5335–5340

Despite high vaccine coverage and protection in the coun-tries that use Hib vaccine, vaccine failures occur, often causedby problems with vaccine delivery or potency. Additionally,a number of studies suggest that variation in the immuneresponse to vaccination is an important determinant of vac-cine efficacy. Siber et al. were the first to demonstrate that theincreased susceptibility of Apache children to invasive Hibdisease directly correlated inversely with antibody responsesto Hib vaccine [2]. Differences in vaccine performance wereobserved in Alaska and Finland even with the use of thesame Hib conjugate vaccine (Hib polysaccharide capsuleconjugated with Diphtheria toxoid vaccine; PRP-D) [3]. Ina different study, post-vaccination PRP antibody levels werethree times higher in Chilean infants than Belgian infants[4]. Furthermore, the Hib carriage rates in vaccinated nativeAlaskan children remained relatively high [5] in contrastto the tremendous reduction in Hib carriage among othervaccinated populations [6–12]. These differences in vaccineresponses between populations are also reflected in host sus-ceptibility to Hib disease. The incidence of invasive Hibdisease is higher in certain ethnic groups (Alaskan natives,African Americans, American Indians, and Australian Abo-rigines) [13]. In contrast, the incidence rate is extremely lowamong the Singaporeans and the Chinese population in HongKong and Mainland China [14].

Whilst population differences in Hib disease and overlap-poImtuFabca

troIaepgomiweiprur

the polysaccharide antigen PRP component of the Hib con-jugate vaccine.

2. Materials and methods

2.1. Study population

This study had the approval of The Gambia Govern-ment/Medical Research Council (MRC) Ethics Committee.Inclusion and exclusion criteria were reported previously[18]. Twins were visited monthly until 5 months of age andvaccinated following the Expanded Programme on Immu-nization. For each vaccine, a single batch was used throughoutthe study and vaccines were given at the same time to eachtwin of a pair. BCG was given intradermally at birth (or at1 month of age if either twin weighed <2.5 kg), oral poliovaccine was given at birth, 1, 2, 3, and 9 months, and hep-atitis B vaccine was given intramuscularly at birth, 2 and 4months. Hib conjugate (ActHIB, Aventis Pasteur) was dilutedin whole-cell diphtheria, tetanus and pertussis vaccine (DTP,Aventis Pasteur) and given intramuscularly at 2, 3 and 4months. Antibody responses to Hib, DT and TT were mea-sured at 5 months of age.

2.2. Hib antibody (anti-PRP IgG) measurement

sAuFoim

2

cow

2

apUnoarbpt

ing vaccines responses will in part relate to varying patternsf disease epidemiology, host genetic factors also play a role.n support of this, siblings of patients with H. influenzaeeningitis have impaired responses to Hib vaccine [15] and

here is a strong correlation between G2m (n) immunoglob-lin allotype and susceptibility to invasive Hib disease [16].urther research demonstrated that the G2m [23] allotype wasssociated with a three-fold increase in IgG subclass2 anti-ody level following Hib polysaccharide vaccination whenompared to responses in individuals who did not carry thisllotype [17].

Immune responses are inherited as complex quantitativeraits with variation resulting from both genetic and envi-onmental factors [18]. In addition to the regulatory effectsf allotypic variants of the gamma-2 heavy chain genes ongG subclasses described above, it is likely that other genesre involved, but the overall contribution of genes versus thenvironment has not been assessed. Twin studies provide aowerful method with which to dissect the relative roles ofenetic and environmental factors in the etiology of disease orther phenotypes [18]. A higher correlation for a trait withinonozygotic (MZ) twin pairs than dizygotic (DZ) twin pairs

ndicates a genetic effect for the trait. A birth cohort of twinsas set up in The Gambia to study the role of genetic and

nvironmental factors in the control of vaccine responses innfants. This study demonstrated that genetic factors play aredominant role in the control of antibody and cytokineesponses to protein antigens [18]. The current study wasndertaken to measure the relative roles of genetic and envi-onmental factors in the control of the antibody response to

Serum anti-PRP IgG concentrations were determined bytandard Hib ELISA methods as described elsewhere [19].ntibody concentrations are measured in �g/ml based on these of an international standard (70 �g/ml) obtained from theood and Drug Administration. The lowest detectable limitf the assay is 0.1 �g/ml and assay coefficient of variations <10%. Serum anti-DT and TT IgG concentrations were

easured by Elisa as previously described [18].

.3. Zygosity determination

The zygosity of same-sex pairs was determined geneti-ally by typing 10 microsatellite markers, as described previ-usly [20]. Twin pairs with identical genotypes for all markersere classified as MZ.

.4. Statistical analysis

Difference in sociodemographic characteristics and in log-rithmic antibody responses between the MZ and DZ twinairs were analysed in Stata 8.2 (Stata Corporation, TX,SA) using random effects logistic regression, adjusting foron-independence of twin pairs. The analysis of continu-us data was based on the natural logarithm of the responsend geometric means are presented. Random effects linearegression was used to assess the association of Hib anti-ody response with sociodemographic characteristics. Intra-air correlations were calculated separately for MZ and DZwins using Pearson’s correlation coefficient. Heritability (the

Y.C. Lee et al. / Vaccine 24 (2006) 5335–5340 5337

genetic contribution to the total phenotypic variation in thepopulation) for Hib antibody response was quantified underan ACE model using Mx path analysis [21]. Briefly, the totalpopulation variance observed for a given phenotype is thesum of genetic and environmental variance, which both canbe further partitioned into two subcomponents; the formerinto additive genetic variance (A) caused by the sum of theaverage effects of all the genes that influence the phenotypeand dominant genetic variance (D) associated with dominantgene action, the latter into common environmental variance(C) (i.e. all non-genetic factors leading to twin similarity)and unique environmental variance (E) specific to each indi-vidual. Mx path analysis fits models to the raw data anddetermines the relative contribution of each of the above fac-tors. We initially fitted a model which allowed for additivegenetic, common environment and unique environment con-tributions to variation (an ACE model). The fit of the datawas compared under the ACE, AE, and CE models, respec-tively. Results show heritability under the final model withpoint estimates and 95% confidence intervals.

3. Results

Two hundred and ninety seven twin pairs were enrolled inthe infant twin study conducted in The Gambia [18]. Of these,zfaictw6t2ps

3a

tea

Table 1Serum anti-PRP antibody concentrations in monozygotic and dizygoticGambian twins after primary immunization

Geometric mean(�g/ml, 95% CI)

Median(�g/ml, 90% range)

MZ (n = 86) 5.53 (3.7–8.2) 6.73 (0.28–48.0)DZ (n = 294) 4.29 (3.5–5.3) 6.49 (0.26–35.4)

Total (n = 378) 4.55 (3.8–5.5) 6.66 (0.27–39.3)

p for difference(MZ vs. DZ) = 0.35

twins are shown in Table 1. The wide range of anti-PRPantibody concentrations confirmed the high inter-individualvariability in antibody responses. No significant differencewas observed in anti-PRP antibody concentrations betweenMZ and DZ twins (p = 0.35).

Significant correlations of anti-PRP IgG concentrationswere observed in both MZ (r = 0.56, p < 0.001) and DZ twinpairs (r = 0.22, p = 0.007). However, the higher correlationcoefficient observed in MZ pairs as compared to DZ pairssuggested genetic control of the response. This was examinedusing the Mx path analysis (Table 2). Under the ACE model,heritability, defined as the proportion of the population vari-ance attributable to additive genetic factors, was estimated at51% (95% CI: 7–66%). Under the same model, the contribu-tion of environmental factors specific to each individual twinof a pair was 49% (95% CI: 34–71%), whereas no significantcontribution was attributable to common environmental fac-tors. The negligible role played by common environmentalfactors in the population variance was further supported bythe fact that the AE model fitted the data as well as the ACEmodel. In contrast, the CE fitted the data significantly worsethan the ACE model. Therefore, the best fitting model forthe anti-PRP IgG responses in Gambian infants was the AEmodel under which heritability was estimated at 51% (95%CI: 32–66%).

3a

trtlc

TM jugate v

M

AAC

0.1 wa

ygosity was determined in 212 pairs and serum was availableor anti-Hib IgG responses in 190 pairs, including 43 MZnd 147 DZ twin pairs. There was no significant differencen zygosity, gender, birth weight, gestational age, or birthentre between those with and without Hib IgG data. Amonghe 190 twin pairs included in this analysis, the mean birtheight was 2.4 kg, and median gestational age was 40 weeks.4 twin pairs were male–male; 49 were female–female, andhe remaining 77 were male–female. Of the 43 MZ twin pairs,4 pairs were male and 19 were female. Of the 147 DZ twinairs, 40 pairs were male, 30 were female and 77 were mixedex.

.1. Antibody responses to Hib conjugate vaccine in MZnd DZ twins

There was no association of Hib antibody response withhe sociodemographic variables and these were not consid-red as potential confounding variables. The geometric meannd the median anti-PRP IgG concentrations of MZ and DZ

able 2x path analysis for heritability estimation of immune response to Hib con

odel Additive geneticfactor A

Common environmentfactor C

CE 0.51 (0.07–0.66) 0 (0–0.28)E 0.51 (0.32–0.66) –E – 0.30 (0.16–0.42)a Defined as −2log likelihood of the model.b Calculated from comparing the fit of the model with the ACE model. p <

.2. Correlation of immune response to Hib, Tetanusnd Diphtheria vaccines

Our previously reported results indicated that genetic fac-ors also play a significant role in the control of early antibodyesponses to TT and DT vaccines that were administered athe same time as the Hib conjugate vaccine in this popu-ation [18]. In order to evaluate whether the same factorsould control the antibody responses to protein and polysac-

accine

Unique environmentfactor E

Goodness of fitstatistica

p-valueb

0.49 (0.34–0.71) 1515.2740.49 (0.34–0.68) 1515.274 10.70 (0.58–0.84) 1520.092 0.03

s considered significant.

5338 Y.C. Lee et al. / Vaccine 24 (2006) 5335–5340

Fig. 1. Correlations between Hib, tetanus and diphtheria vaccine responsesin Gambian twins. Antibody responses were measured at 5 months of agein 370 twins (80 MZ and 290 DZ) immunised at 2, 3 and 4 months.

charide antigens, we studied whether these responses werecorrelated in our study population. A high correlation wasobserved between DT and TT responses (r = 0.62, p < 0.001)(Fig. 1(a)), as reported previously [18]. In contrast, signifi-cant but weaker correlations were observed between anti-Hiband anti-DT (r = 0.23, p < 0.001) (Fig. 1(b)) or anti-Hib andanti-TT (r = 0.26, p = 0.001) (Fig. 1(c)) IgG concentrations.

4. Discussion

This study quantified the relative contribution of geneticand environmental factors to the variation in the antibodyresponse to the Hib conjugate vaccine in early life. Anti-body response to Hib vaccine was highly variable in Gambianinfants. We observed that 51% of the population variance wasattributable to genetic factors. Environmental factors specificto individual twins accounted for the remaining 49% of thevariance. These results indicate that the antibody responseto Hib vaccine is highly heritable in the Gambian popula-tion. Similar results were obtained with immune responsesto protein antigens, including TT and DT [18].

Low but significant correlations were observed betweenHib and TT or DT responses. As observed in other stud-ies, the correlation with TT was somewhat higher than withDT, a phenomenon that may be attributable to the fact thatthe Hib vaccine was conjugated with TT [22–26]. Impor-tantly, however, TT responses were more correlated with DTresponses than with Hib responses. These results suggest thatthe factors which control early antibody responses to diverseprotein antigens are in part distinct from those controllingthe response to polysaccharide antigens. The identificationof the genes involved should allow us to determine whetherantibody responses to protein and polysaccharide antigensinvolve similar or distinct genes.

iclNgiiHhHAgHfamHo

olispiccea

Our results have important implication for the understand-ng of the variable incidence rates in invasive disease, Hibarriage and vaccine performance among different popu-ations. The incidence of invasive Hib disease among theavajo and White Mountain Apache children is still 20 timesreater than that observed among children of the same agen the general US population despite the decline after themplementation of Hib vaccine [27]. Also, relatively highib carriage rates are observed in Alaskan natives despiteigh rates of vaccine coverage [5] and efficacy of the sameib conjugate vaccine is different in Finland and Alaska [3].lthough these differences may involve environmental andenetic factors, our observation that antibody responses toib vaccine are genetically controlled suggests that genetic

actors play an important role. When exposed to Hib bacteria,poorer antibody response in a subset of human populationsight potentially increase Hib carriage or the risk of invasiveib disease. Further studies are needed to determine the rolef genetic factors in the control of Hib carriage.

Our results also suggest a role for genetic factors in theccurrence of vaccine failures in the UK population. Fol-owing 13 years of active Hib surveillance and Hib routinemmunization regime in the UK, an increase in cases of inva-ive Hib disease was noted after 1998 from a rate of 0.65 caseser 100,000 children [28] to 4.6 cases per 100,000 childrenn 2002 [29]. The earlier studies had shown very high vac-ine efficacy of 98% and excellent immunogenicity in healthyhildren [1,30,31] as well as in children with underlying dis-ase (e.g. sickle cell disease [32], congenital asplenia [33]nd those with bone marrow transplants [34]), an interaction

Y.C. Lee et al. / Vaccine 24 (2006) 5335–5340 5339

of the Hib vaccine with a newly introduced acellular pertus-sis vaccine which attenuated the immune response to Hib isbelieve to account for much of the increase in the rate of dis-ease after 1999 [35]. The dosing schedule used in the UKmay also have played a part. The majority of the countriesthat use Hib vaccine give three priming doses in infancy andthen offer a booster dose of Hib in the second year of life.However, Hib vaccine was introduced in the UK without abooster dose, so that protection relied on the primary immuneresponse only [36]. Most of these cases were children withHib vaccine failure, occurring in the first 4 years of life. Nev-ertheless, despite these environmental stresses applied to theimmune response, since all infants were primed the sameway, the occurrence of vaccine failure highlights the pres-ence of a special subset of the population with susceptibilityto Hib disease despite immunization. Previous studies haveshown that convalescent children with Hib vaccine failurehad an anamnestic antibody response to PRP indicating thatthey had been primed with the vaccine [37,38]. Although werecognise that caution should be exercised in extrapolatingfrom our African data to a Caucasian population, we pro-pose that vaccine failures may be related to poor antibodyresponses to the Hib vaccine and that our data suggest thatgenetic factors may be involved. As samples were collectedduring the early phase of the vaccine response, we could notexamine the role of genetic factors in the persistence andapdc

ictsav

A

AAfKt

iWthhGfP

meetings as well as financial support to conduct researchprojects on behalf of the University of Geneva.

References

[1] Watt JP, Levine OS, Santosham M. Global reduction of Hib disease:what are the next steps? Proceedings of the meeting Scottsdale, Ari-zona. J Pediatr 2003;143(Suppl. 6):S163–87.

[2] Siber GR, Santosham M, Reid GR, Thompson C, Almeido-Hill J,Morell A, et al. Impaired antibody response to Haemophilus influen-zae type b polysaccharide and low IgG2 and IgG4 concentrations inApache children. N Engl J Med 1990;323(20):1387–92.

[3] Heath PT. Haemophilus influenzae type b conjugate vaccines:a review of efficacy data. Pediatr Infect Dis J 1998;17(Suppl.9):S117–22.

[4] Hetherington SV, Lepow ML. Correlation between antibody affin-ity and serum bactericidal activity in infants. J Infect Dis1992;165(4):753–6.

[5] Galil K, Singleton R, Levine OS, Fitzgerald MA, Bulkow L, GettyM, et al. Reemergence of invasive Haemophilus influenzae type bdisease in a well-vaccinated population in remote Alaska. J InfectDis 1999;179(1):101–6.

[6] Mohle-Boetani JC, Ajello G, Breneman E, Deaver KA, Harvey C,Plikaytis BD, et al. Carriage of Haemophilus influenzae type b inchildren after widespread vaccination with conjugate Haemophilusinfluenzae type b vaccines. Pediatr Infect Dis J 1993;12(7):589–93.

[7] Murphy TV, Pastor P, Medley F, Osterholm MT, Granoff DM.Decreased Haemophilus colonization in children vaccinated withHaemophilus influenzae type b conjugate vaccine. J Pediatr

[

[

[

[

[

[

[

vidity maturation of anti-PRP antibodies. Since waning ofrotective antibody is likely to lead to susceptibility to Hibisease, genetic studies that evaluate this important questionould allow prediction of those who might be at risk.

We conclude that genetic factors play an important rolen the control of antibody response to Hib conjugate vac-ine in Gambian infants. Further work is needed to identifyhe specific genes involved. This will lead to better under-tanding of the early immune interaction with polysaccharidentigen and thus allow for the development of improvedaccines.

cknowledgements

This project was funded by the University of Melbourne,ustralia and conducted at the University of Oxford, UK.rnaud Marchant is a research associate of the National Fund

or Scientific Research, Belgium. The authors thank Dominicelly and Elizabeth Clutterbuck for assistance in the labora-

ory methods and analysis.Conflict of interest statement: AJP has conducted clin-

cal trials on behalf of Oxford University, sponsored byyeth Vaccines, GlaxoSmithKline vaccines, Sanofi Aven-

is, Sanofi Pasteur MSD and Chiron vaccines. AJP does notold any paid consultancies with vaccine manufacturers. AMas served as consultant and received research support fromlaxoSmithKline Biologicals. CAS has received honoraria

rom GlaxoSmithKline, Wyeth, Sanofi Aventis and Sanofiasteur MSD to participate to scientific advisory boards and

1993;122(4):517–23.[8] Takala AK, Eskola J, Leinonen M, Kayhty H, Nissinen A, Pekka-

nen E, et al. Reduction of oropharyngeal carriage of Haemophilusinfluenzae type b (Hib) in children immunized with an Hib conjugatevaccine. J Infect Dis 1991;164(5):982–6.

[9] Barbour ML, Mayon-White RT, Coles C, Crook DW, Moxon ER.The impact of conjugate vaccine on carriage of Haemophilus influen-zae type b. J Infect Dis 1995;171(1):93–8.

10] Adegbola RA, Mulholland EK, Secka O, Jaffar S, Greenwood BM.Vaccination with a Haemophilus influenzae type b conjugate vac-cine reduces oropharyngeal carriage of H. influenzae type b amongGambian children. J Infect Dis 1998;177(6):1758–61.

11] Forleo-Neto E, de Oliveira CF, Maluf EM, Bataglin C, AraujoJM, Kunz Jr LF, et al. Decreased point prevalence of Haemophilusinfluenzae type b (Hib) oropharyngeal colonization by mass immu-nization of Brazilian children less than 5 years old with Hib polyribo-sylribitol phosphate polysaccharide-tetanus toxoid conjugate vaccinein combination with diphtheria–tetanus toxoids–pertussis vaccine. JInfect Dis 1999;180(4):1153–8.

12] Hargreaves RM, Slack MP, Howard AJ, Anderson E, Ramsay ME.Changing patterns of invasive Haemophilus influenzae disease inEngland and Wales after introduction of the Hib vaccination pro-gramme. BMJ 1996;312(7024):160–1.

13] Parke Jr JC, Schneerson R, Robbins JB. The attack rate, age inci-dence, racial distribution, and case fatality rate of Haemophilusinfluenzae type b meningitis in Mecklenbury County, North Car-olina. J Pediatr 1972;81(4):765–9.

14] Lolekha S, Cooksley G, Chan V, Isahak I, Ismael S, John J, et al. Areview of Hib epidemiology in Asia. Southeast Asian J Trop MedPublic Health 2000;31(4):650–7.

15] Granoff DM, Squires JE, Munson Jr RS, Suarez B. Siblingsof patients with Haemophilus meningitis have impaired anti-capsular antibody responses to Haemophilus vaccine. J Pediatr1983;103(2):185–91.

16] Ambrosino DM, Schiffman G, Gotschlich EC, Schur PH, Rosen-berg GA, DeLange GG, et al. Correlation between G2m (n)

5340 Y.C. Lee et al. / Vaccine 24 (2006) 5335–5340

immunoglobulin allotype and human antibody response and sus-ceptibility to polysaccharide encapsulated bacteria. J Clin Invest1985;75(6):1935–42.

[17] Granoff DM, Suarez BK, Pandey JP, Shackelford PG. Genes asso-ciated with the G2m (23) immunoglobulin allotype regulate the IgGsubclass responses to Haemophilus influenzae type b polysaccharidevaccine. J Infect Dis 1988;157(6):1142–9.

[18] Newport MJ, Goetghebuer T, Weiss HA, Whittle H, Siegrist CA,Marchant A. Genetic regulation of immune responses to vaccines inearly life. Genes Immun 2004;5(2):122–9.

[19] Phipps DC, West J, Eby R, Koster M, Madore DV, Quataert SA. AnELISA employing a Haemophilus influenzae type b oligosaccharide–human serum albumin conjugate correlates with the radioantigenbinding assay. J Immunol Methods 1990;135(1/2):121–8.

[20] Becker A, Busjahn A, Faulhaber HD, Bahring S, Robertson J,Schuster H, et al. Twin zygosity. Automated determination withmicrosatellites. J Reprod Med 1997;42(5):260–6.

[21] Neale MC, Miller MB. The use of likelihood-based confidence inter-vals in genetic models. Behav Genet 1997;27(2):113–20.

[22] Bell F, Martin A, Blondeau C, Thornton C, Chaplais J, Finn A.Combined diphtheria, tetanus, pertussis, and Haemophilus influen-zae type b vaccines for primary immunization. Arch Dis Child1996;75(4):298–303.

[23] Dagan R, Eskola J, Leclerc C, Leroy O. Reduced responseto multiple vaccines sharing common protein epitopes that areadministered simultaneously to infants. Infect Immun 1998;66(5):2093–8.

[24] Finn A, Blondeau C, Bell F. Haemophilus influenzae type B (Hib)antibody responses in children given diphtheria–tetanus–acellularpertussis–Hib combination vaccines. J Infect Dis 2000;181(6):2117–8.

[

[

[27] Bisgard KM, Kao A, Leake J, Strebel PM, Perkins BA, WhartonM. Haemophilus influenzae invasive disease in the United States,1994–1995: near disappearance of a vaccine-preventable childhooddisease. Emerg Infect Dis 1998;4(2):229–37.

[28] Heath PT, McVernon J. The UK Hib vaccine experience. Arch DisChild 2002;86(6):396–9.

[29] Ramsay ME, McVernon J, Andrews NJ, Heath PT, Slack MP.Estimating Haemophilus influenzae type b vaccine effectiveness inEngland and Wales by use of the screening method. J Infect Dis2003;188(4):481–5.

[30] Heath PT, Booy R, Azzopardi HJ, Slack MP, Bowen-Morris J,Griffiths H, et al. Antibody concentration and clinical protectionafter Hib conjugate vaccination in the United Kingdom. JAMA2000;284(18):2334–40.

[31] Kelly DF, Moxon ER, Pollard AJ. Haemophilus influenzae type bconjugate vaccines. Immunology 2004;113(2):163–74.

[32] Goldblatt D, Johnson M, Evans J. Antibody responses toHaemophilus influenzae type b conjugate vaccine in sickle cell dis-ease. Arch Dis Child 1996;75(2):159–61.

[33] Webber SA, Sandor GG, Patterson MW, Mitchell LA, Scheifele D,Ochnio JJ, et al. Immunogenicity of Haemophilus influenzae type bconjugate vaccine in children with congenital asplenia. J Infect Dis1993;167(5):1210–2.

[34] Guinan EC, Molrine DC, Antin JH, Lee MC, Weinstein HJ, Sal-lan SE, et al. Polysaccharide conjugate vaccine responses in bonemarrow transplant patients. Transplantation 1994;57(5):677–84.

[35] McVernon J, Andrews N, Slack MP, Ramsay ME. Risk of vaccinefailure after Haemophilus influenzae type b (Hib) combination vac-cines with acellular pertussis. Lancet 2003;361(9368):1521–3.

[36] McVernon J, Mitchison NA, Moxon ER. T helper cells and effi-cacy of Haemophilus influenzae type b conjugate vaccination. Lancet

[

[

25] Goldblatt D, Richmond P, Millard E, Thornton C, Miller E.The induction of immunologic memory after vaccination withHaemophilus influenzae type b conjugate and acellular pertussis-containing diphtheria, tetanus, and pertussis vaccine combination. JInfect Dis 1999;180(2):538–41.

26] Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of sero-logical memory by polyclonal activation of human memory B cells.Science 2002;298(5601):2199–202.

Infect Dis 2004;4(1):40–3.37] McVernon J, Johnson PD, Pollard AJ, Slack MP, Moxon ER.

Immunologic memory in Haemophilus influenzae type b conjugatevaccine failure. Arch Dis Child 2003;88(5):379–83.

38] Heath PT, Booy R, Griffiths H, Clutterbuck E, Azzopardi HJ, SlackMP, et al. Clinical and immunological risk factors associated withHaemophilus influenzae type b conjugate vaccine failure in child-hood. Clin Infect Dis 2000;31(4):973–80.