Vaccine 22 (2004) Old Road, Headington, Oxford OX3 7LJ, UK b Aventis Pasteur MSD Ltd., Mallards Reach, Maidenhead, Berkshire, UK

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1 Vaccine 22 (2004) Immunogenicity and safety of a low-dose diphtheria, tetanus and acellular pertussis combination vaccine with either inactivated or oral polio vaccine as a pre-school booster in UK children C.L. Collins a, P. Salt a, N. McCarthy a, T. Chantler a, L. Lane a, F. Hemme b, L. Diggle a, J. Buttery a, N.R.E. Kitchin b, E.R. Moxon a, A.J. Pollard a, a Oxford Vaccine Group, Centre for Clinical Vaccinology & Tropical Medicine, University of Oxford, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, UK b Aventis Pasteur MSD Ltd., Mallards Reach, Maidenhead, Berkshire, UK Received 26 January 2004; accepted 26 April 2004 Abstract This open, randomised controlled trial studied the immunogenicity and reactogenicity of two combined low-dose diphtheria, tetanus and acellular pertussis vaccines (Td5aP-IPV, REPEVAX TM, Aventis Pasteur MSD; and Td5aP, COVAXIS TM, Aventis Pasteur MSD + OPV, GlaxoSmithKline) in comparison with a standard dose diphtheria pre-school booster vaccine (DT2aP-IPV, TETRAVAC TM, Aventis Pasteur MSD) in a population of year-old children administered concomitantly with measles, mumps and rubella vaccine (M-M-R TM II, Aventis Pasteur MSD). A linked sub-study aimed to evaluate the immunogenicity and reactogenicity of Td5aP-IPV in a population of younger children, aged years. This study demonstrated non-inferiority of seroprotection rates for diphtheria and tetanus for the study vaccines and comparable immunogenicity for pertussis and polio components of the vaccines. Reactogenicity was similar for all three vaccines. The study vaccines containing low-dose diphtheria antigen (Td5aP-IPV and Td5aP + OPV) are immunogenic and have acceptable reactogenicity for use as a pre-school booster vaccine administered concomitantly with MMR Elsevier Ltd. All rights reserved. Keywords: Low dose diphtheria vaccine; Pre-school booster; Immunogenicity and safety 1. Introduction The introduction of diphtheria toxoid vaccines in the UK in the 1940s was associated with a dramatic decrease in mortality and morbidity caused by infection with Corynebacterium diphtheriae [1]. However, the recent outbreaks of diphtheria in several of the independent states of the former Soviet Union has demonstrated the need for an ongoing effective and acceptable vaccination programme [2]. In most industrialised nations, diphtheria prevention programmes include up to three booster doses of diphtheria toxoid after infancy, with a view to providing sustained protection. Corresponding author. Tel.: ; fax: address: andrew.pollard@paediatrics.ox.ac.uk (A.J. Pollard). Currently, children in the UK receive their first booster dose of diphtheria toxoid in combination with tetanus and pertussis antigens (and oral polio vaccine; DTaP + OPV + MMR) before school entry and at least 3 years after completion of the infant 2-, 3- and 4-month primary course [3] ( Younger age at school entry and increasing use of day care nurseries in the UK have been associated with a trend amongst General Practitioners to give the pre-school booster earlier in the recommended age range of years and doses may occasionally be given during the first half of the fourth year of life. It is clearly important to minimise the reactogenicity of booster doses of these vaccines if high levels of vaccine coverage are to be maintained. The inclusion of multiple booster X/$ see front matter 2004 Elsevier Ltd. All rights reserved. doi: /j.vaccine

2 C.L. Collins et al. / Vaccine 22 (2004) doses of diphtheria antigen during childhood has raised concerns over local reactions (mainly erythema and induration). As a result, low-dose diphtheria vaccine is recommended in the UK for children over the age of 10 years who receive the vaccine as part of the adolescent booster but no such recommendations currently exist for booster doses administered earlier in childhood [3]. The inclusion of the mutant diphtheria toxoid (CRM197) containing group C meningococcal conjugate vaccines into the routine UK immunisation schedule in 1999 has also increased the amount of diphtheria antigen exposure during early infancy. Furthermore, there is a possibility that a pneumococcal conjugate vaccine containing CRM197 will be included in the UK vaccine schedule in the future. It is not known whether these vaccines will have a significant impact on reactogenicity of diphtheria toxoid-containing vaccines administered through childhood or whether an improved safety profile might be expected by replacement of standard dose diphtheria vaccine with a low-dose diphtheria vaccine. Acellular pertussis vaccine was added to the pre-school booster in the UK in 2001 to enhance population immunity against pertussis in the school population and reduce secondary cases among infants too young to have been vaccinated (i.e. <2 months of age). Acellular pertussis vaccine was used in view of the decreased reactogenicity of this vaccine in contrast to whole cell pertussis vaccine, which is included in the infant immunisation schedule in the UK. Inactivated polio vaccine (IPV) is a potential candidate for inclusion into the UK vaccine schedule as the relative importance of vaccine-associated paralysis (VAPP) [4] following administration of live attenuated oral polio (OPV) has increased with the decrease in the number of cases of paralytic polio caused by the wild virus. In this study, the immunogenicity and reactogenicity of vaccines containing low-dose diphtheria toxoid that might be used as a pre-school booster in the UK was examined in two related studies. The main study involved a population of children aged years and aimed to show non-inferiority for diphtheria and tetanus immunogenicity using two vaccines containing a low-dose diphtheria toxoid in comparison with a standard dose diphtheria vaccine. Immunogenicity of pertussis and polio components of these vaccines and their reactogenicity were also described. A sub-study aimed to extend evaluation of a low-dose diphtheria vaccine to children aged years of age. 2. Methods 2.1. Vaccines The main study was an open randomised controlled non-inferiority trial. The two investigational vaccines were combined aluminium phosphate adsorbed tetanus toxoid (Lf/dose; 20 IU/dose), low-dose diphtheria toxoid (Lf/dose; 2 IU/dose), five-component acellular pertussis (purified pertussis toxoid (PT), 2.5 g; purified filamentous haemagglutinin (FHA), 5 g; purified fimbrial agglutinogens 2+3(FIM), 5 g; purified pertactin (PRN), 3 g; inactivated poliomyelitis virus (types 1 3) vaccine (Td5aP-IPV, REPEVAX TM, Aventis Pasteur MSD) and combined aluminium phosphate adsorbed tetanus toxoid (Lf/dose; 20 IU/dose), low-dose diphtheria toxoid (Lf/dose; 2 IU/dose) and five-component (as for REPEVAX TM ) acellular pertussis vaccine (Td5aP, COVAXIS TM, Aventis Pasteur MSD). The control vaccine contained combined aluminium hydroxide adsorbed tetanus toxoid ( 40 IU/dose), standard dose diphtheria toxoid ( 30 IU/dose), two-component acellular pertussis (purified PT, 25 g; and purified FHA, 25 g) and inactivated poliomyelitis vaccine (DT2aP-IPV, TETRAVAC TM, Aventis Pasteur MSD). Two concomitant vaccines were used in the trial. Oral poliomyelitis vaccine (OPV, GlaxoSmithKline) was given to those receiving Td5aP and measles, mumps and rubella vaccine (live attenuated, M-M-R TM II, Aventis Pasteur MSD) was administered to all subjects. In the sub-study of younger children, all received Td5aP- IPV and M-M-R TM II Subjects Parents with children in Oxfordshire and the town of Milton Keynes aged between 3.5 and 5 years were approached to participate in the main study. The sub-study included children aged between 3 and 3.5 years of age only. Both studies were approved by the Oxfordshire Research and Ethics Committee (CO1.83) and Milton Keynes Ethics Committee (MKLREC\14\02) and were conducted in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines. In order to be eligible for entry to the trial, all children were to have had their primary immunisations completed according to the UK schedule at the time of vaccination. These included three doses of diphtheria, tetanus, whole cell pertussis, Haemophilus influenzae type b and OPV at least 3 years (2.5 years in the sub-study) previously. In addition, all had received one dose of measles, mumps and rubella combined vaccine (MMR). In the main study, children had received a single dose of meningococcal serogroup C (Men C) conjugate vaccine as part of the nationwide catch-up campaign which accompanied this vaccine s addition to the routine UK immunisation schedule in October The sub-study included only children whose full primary infant course included three doses of Men C vaccine in infancy given concomitantly with the DTwP vaccine. Exclusion criteria were: prior clinical or bacteriological diagnosis of diphtheria, tetanus, pertussis or poliomyelitis; immunosuppression; receipt of any vaccine in the previous 4 weeks or immunoglobulins within the previous 3 months; allergy to vaccine components; and parental refusal to consent to the study protocol including concomitant administration of MMR.

3 4264 C.L. Collins et al. / Vaccine 22 (2004) Study design The main study was an open, randomised, controlled, phase II study of the immunogenicity and safety of combined adsorbed tetanus, low-dose diphtheria, five-component acellular pertussis and inactivated poliomyelitis vaccine (Td5aP- IPV), concomitant combined adsorbed tetanus, low-dose diphtheria and five-component acellular pertussis vaccine (Td5aP) and oral poliomyelitis vaccine (OPV) and combined adsorbed diphtheria, tetanus, two-component acellular pertussis and inactivated poliomyelitis vaccine (DT2aP-IPV) given to healthy UK children a minimum of 3 years after priming with diphtheria, tetanus and whole cell pertussis (DTwP) vaccine at 2, 3 and 4 months of age. In this study, the primary endpoint was demonstration that the immune response to both diphtheria and tetanus toxoids 4 6 weeks after receipt of Td5aP-IPV or Td5aP + OPV is non-inferior to that after DT2aP-IPV. The study also aimed to describe the immune response to pertussis (PT, FHA, FIM and PRN) and poliomyelitis (types 1 3) antigens 4 6 weeks after receipt of study vaccines and to describe the reactogenicity (in terms of local and systemic adverse event profile) of each of the vaccines. The sub-study was an open, uncontrolled, phase IV study of the immunogenicity and safety of combined adsorbed tetanus, low-dose diphtheria, five-component acellular pertussis and inactivated poliomyelitis vaccine (Td5aP-IPV) given to healthy UK children a minimum of 2.5 years after priming with diphtheria, tetanus and whole cell pertussis vaccine at 2, 3 and 4 months of age. This study aimed to describe the immune response to the antigens contained in Td5aP-IPV 4 6 weeks post-vaccination and to demonstrate non-inferiority in the response to Td5aP-IPV in children aged years compared to the older children in the main study receiving this vaccine. Secondary endpoints were comparison of the immune response to the other antigens contained in Td5aP-IPV 4 6 weeks post-vaccination with the data from the older children (in the main study), description of the reactogenicity (in terms of local and systemic adverse event profile) of Td5aP-IPV and comparison of the reactogenicity of Td5aP-IPV with historical data in older children in the main study Study procedures Subjects in the main study were allocated to receive one of the three vaccines described above by computer generated randomisation, grouped in blocks of three. Parents gave their informed consent in writing at the first study visit. Subjects were vaccinated intramuscularly into the deltoid muscle using a 16 mm, 25-gauge needle. The diphtheriacontaining study vaccine was administered into the right deltoid and MMR into the left. Subjects were observed for min post-immunisation for immediate adverse events. Parents were asked to complete a reactogenicity diary for 7 days following immunisation. Blood samples were taken pre-vaccination and at the second visit days later Serological assays Widely accepted correlates of protection for diphtheria and tetanus antitoxin are 0.1 IU/ml and 0.1 EU/ml, respectively [5,6], although there are well-known difficulties in standardizing the assays. Anti-diphtheria antitoxin was measured by microneutralisation using a micrometabolic inhibition test with reference standards (the WHO International Standard for Diphtheria Antitoxin, First International Standard Preparation, National Institute for Biological Standards and Control (NIBSC), UK). Anti-tetanus antitoxin was measured by enzyme-linked immunoassay using an in-house reference standard to quantitate tetanus antitoxin. Antibodies to PT, FHA, FIM and pertactin were measured by enzymelinked immunoassay using in-house reference standards. Antibodies to poliomyelitis virus types 1 3 were measured by a neutralisation assay using in-house reference standards calibrated to the First International Standard Anti-Poliovirus Serum, types 1 3 (NIBSC, UK), established by the Expert Committee on Biological Standardization, WHO. All of the assays were performed in the clinical serology laboratories of Aventis Pasteur, Toronto, Canada, with laboratory staff unaware of subject assignment. Serum samples with levels less than the detection limit were assigned a value of one-half the lower detection limit when calculating geometric means. 3. Statistical analysis and sample size 3.1. Sample size for main study The sample size was based upon the proportion of subjects expected to achieve seroprotective anti-diphtheria and anti-tetanus antitoxin titres, defined as greater than or equal to 0.1 IU/ml or 0.1 EU/ml, respectively. With a sample size of 300, this study had 81% overall power to demonstrate non-inferiority of the two investigational groups assuming a reference seroprotection rate of 99%, with a clinical equivalence range of 5% and allowing for a 14% non-evaluable rate among participants Statistical analysis Demographic characteristics and baseline levels of anti-diphtheria and anti-tetanus toxoid antibodies were described for each group. The primary endpoint was comparison of the percentage of subjects in each group with protective anti-diphtheria antitoxin levels 0.1 IU/ml and anti-tetanus antitoxin levels 0.1 EU/ml post-immunisation. Groups were compared by estimation of differences in the percentage exceeding these levels of antibody, with 90% confidence interval (CI), an upper CI of less than 5% indicating non-inferiority, in line with the criteria of non-inferiority applied in this study. As the number of subjects was calculated based on 90% CI, those confidence intervals are reported in the text. Nevertheless, 95% confidence intervals were also calculated and

4 C.L. Collins et al. / Vaccine 22 (2004) lead to the same conclusion for Td5aP-IPV (REPEVAX TM ) and are presented in the tables. Intention to treat analyses are tabulated for all outcomes. The trial protocol specified perprotocol analysis to assess non-inferiority for the primary outcomes and these per-protocol results are reported for primary outcomes in Section 4. For all other comparisons of proportions between groups Fisher s exact test [7] was used. Geometric mean titres (GMT) of anti-diphtheria, anti-tetanus, polio (types 1 3) and pertussis (PT, FHA, FIM and PRN) antibodies post-immunisation were calculated with 95% confidence intervals and compared using t-tests. Analysis was repeated with rank-sum tests to assess the effect of the assumption of normally distributed data. These non-parametric analyses yielded similar results and only parametric results are presented. To investigate reactogenicity, local and systemic reactions occurring during the 7 days following vaccination were described. The proportion in each group reporting local (pain, erythema and swelling) or systemic (pyrexia, diarrhoea, rash, swollen joints, tiredness, vomiting) side effects was calculated and compared between groups using Fisher s exact test [7] Sub-study children aged years of age Descriptive data were analysed by calculating means, medians or proportions where appropriate, 95% CIs were provided for all descriptive estimates and a 5% significance level was used throughout. Descriptive comparisons were made with data from the older group of children. Comparisons were made using 90% CIs of the difference of the seroprotection rates between the 100 children aged years who received Td5aP-IPV and the 50 children aged years who received Td5aP-IPV. 4. Results 4.1. Main study Study group characteristics and compliance Three hundred subjects aged years were enrolled into the main study and immunised. Forty-five percent of the children were male and the mean age was 3.9 years. A total of nine subjects deviated from protocol. Lower numbers of participants in individual analyses are due to insufficient serum being available for all analyses from all patients (Table 1) Immunogenicity All but one of the subjects for whom data was available achieved protective antibody titres as defined by the primary outcome for the study for both diphtheria and tetanus (Table 2). Comparison of each low-dose diphtheria vaccine group against the standard dose group showed a difference (proportion protected by standard vaccine minus proportion Table 1 Subject flowchart Main study Sub-study Age groups of subjects (years) Number approached Total number of replies Number of positive replies Number excluded >1 Dose Men C (3.5 5 only) n =65 No MMR n =9 n =3 Different infant vaccination schedule n =8 n =1 Out of age range n =1 Already had pre-school booster n = 139 No pre-vaccination serum obtained n =5 n =1 Subject on high dose steroids n =1 Replied after recruitment closed n =14 n =63 Number randomised by group 100 (Group 1) 100 (Group 2) 100 (Group 3) 50 (Group 4) Mean age in years (range) 4.0 ( ) 3.9 ( ) 3.9 ( ) 3.2 ( ) Percentage male Vaccine given Td5aP-IPV Td5aP + OPV DT2aP-IPV Td5aP-IPV Protocol violations >1 Dose of Men C(n =1) Second visit out of protocol time >1 Dose of Men C (n = 1); no post-vaccination blood No post-vaccination blood sample (n =1) window (n =1) sample (n = 6); total (n =7) Pre-vaccination serum obtained Post-vaccination serum obtained a Number contributing to diphtheria and b 49 tetanus immunogenicity data Number contributing to safety data a Serum insufficient for all planned tests in some cases leading to reduced sample sizes as indicated in later tables. b Ninety-two contributed to immunogenicity data for tetanus and 91 for diphtheria.

5 4266 C.L. Collins et al. / Vaccine 22 (2004) Table 2 Pre- and post-vaccination geometric mean titres and proportion with a protective antibody level for diphtheria and tetanus, for participants with available follow up serology, by study group Main study (3.5 5 years) a Sub-study (3 3.5 years) a DT2aP-IPV Td5aP + OPV Td5aP-IPV Td5aP-IPV Diphtheria n =91 b n = 100 n =98 n =49 Pre-vaccination GMT IU/ml [95% CI] 0.50 [ ] 0.41 [ ] 0.44 [ ] 0.03 [ ] Percent 0.1 IU/ml pre-vaccination [95% CI] 78 [68 86] 63 [53 72] 67 [57 76] 8 [2 20] Post-vaccination GMT IU/ml [95% CI] 23 [16 34] 17 [12 24] 11 c [8 16] 7 [5 10] Percent 0.1 IU/ml post-vaccination [95% CI] 100 [96 100] 99 [95 100] 100 [96 100] 100 [93 100] Tetanus n =92 n = 100 n =98 n =49 Pre-vaccination GMT EU/ml [95% CI] 0.26 [ ] 0.25 [ ] 0.27 [ ] 0.19 [ ] Percent 0.1 EU/ml pre-vaccination [95% CI] 89 [81 95] 85 [76 91] 89 [81 94] 80 [66 90] Post-vaccination GMT EU/ml [95% CI] 15 [12 18] 30 c [25 35] 20 c [17 23] 19 [15 24] Percent 0.1 EU/ml post-vaccination [95% CI] 100 [96 100] 100 [96 100] 100 [96 100] 100 [93 100] a Age groups of subjects. b n = 90 for baseline diphtheria serology as no result available for one subject. c Geometric mean titres significantly different (P < 0.01) to those for DT2aP-IPV. by low-dose vaccine) of 0.0% [90% CI: 2.9 to +2.7%] for Td5aP-IPV and 1.0% [90% CI: 1.9 to +4.4%] for Td5aP + OPV for diphtheria. For tetanus, the differences were 0.0% [90% CI: 2.9 to +2.7%] for Td5aP-IPV and 0.0% [95% CI: 2.9 to +2.6%] for Td5aP + OPV demonstrating non-inferiority for both trial vaccines for antibodies against tetanus and diphtheria toxoids. Both per-protocol and intention to treat analysis yielded identical point estimates and confidence intervals. Diphtheria antitoxin geometric mean titres were lower in the sera of children who received Td5aP-IPV when compared with Td5aP + OPV or DT2aP-IPV. Tetanus antitoxin GMTs in the Td5aP-IPV and Td5aP + OPV were significantly higher, despite having lower doses of tetanus toxoid than DT2aP-IPV (Table 2). The immunogenicity of the two five-component pertussis vaccines was very similar for the pertussis antigens assessed in this trial. Geometric mean antibody titres against filamentous haemagglutinin were somewhat higher in the two-component group. Geometric mean antibody titres against pertussis toxoid were lower in the two-component group than either of the five-component vaccines (P < 0.01) although the dose is 10 times greater in the two-component vaccine (Table 3). Polio immunogenicity as measured by serum antibody level was substantially lower among subjects receiving the OPV compared to both IPV groups (P < 0.01) (Table 3) Reactogenicity Local and systemic reactions were similar in the three groups except for a significant excess of local erythema and of rash in Td5aP + OPV compared to the control vaccine, DT2aP-IPV (Table 4). Table 3 Post-vaccination geometric mean titres of pertussis antibodies and reciprocal dilutions for polio antibodies, by study group Main study (3.5 5 years) a Sub-study (3 3.5 years) a DT2aP-IPV Td5aP-OPV Td5aP-IPV Td5aP-IPV Post-vaccination n =93 n = 100 n = 100 n =49 Pertussis antigens Geometric mean titres (EU/ml) [95% confidence intervals] PT 97 [77 123] 156 [ ] 159 [ ] 220 [ ] FHA 187 [ ] 161 [ ] 123 [98 154] 99 [72 136] FIM 38 [29 50] 997 [ ] 826 [ ] 472 [ ] PRN 7 [6 9] 338 [ ] 239 [ ] 149 [ ] Post-vaccination n =93 n = 100 n =99 n =49 Polio types Reciprocal dilutions [95% confidence interval] Polio [ ] 2077 [ ] [ ] [ ] Polio [ ] [ ] [ ] [ ] Polio [ ],b 350 [ ] [ ],c [ ] a Age groups of subjects. b n = 92. c n = 97. Significantly higher than DT2aP (P < 0.01). Significantly higher than in the OPV group (P < 0.01).

6 C.L. Collins et al. / Vaccine 22 (2004) Table 4 Local and systemic reactions 1 7 days post-vaccination, by study group Main study (3.5 5 years) a Sub-study (3 3.5 years) a DT2aP-IPV Td5aP + OPV Td5aP-IPV Td5aP-IPV Number in group Percent with reported reaction [95% confidence interval] Erythema 39 [29 49] 61 [51 71] b 47 [37 57] 52 [37 66] Pain 65 [55 74] 71 [61 80] 72 [62 81] 70 [55 82] Swelling 34 [25 44] 40 [30 50] 37 [28 47] 28 [16 42] Pyrexia 16 [9 25] 13 [7 21] 15 [9 24] 8 [2 19] Rash 8 [4 15] 21 [13 30] c 10 [5 18] 6 [1 17] a Age groups of subjects. b Erythema Td5aP + OPV vs. DT2aP-IPV (P = 0.003). c Rash Td5aP + OPV vs. DT2aP-IPV (P = 0.015) Adverse reactions Adverse events which required a medical consultation and serious adverse events were assessed in each group. There were no serious adverse events and no subjects withdrew due to adverse events Sub-study in younger children Baseline characteristics and compliance Fifty subjects aged years were enrolled into the study and immunised with Td5aP-IPV. Fifty-four percent of the children were male and the mean age was 3.2 years. There was no post-vaccination blood sample available from one subject (Table 1). The pre-vaccination GMT for anti-diphtheria toxoid was substantially lower in this group when compared with the year olds (see Table 2) Immunogenicity All 49 children with available data met the primary immunogenicity outcome criteria for protection against both diphtheria and tetanus. Comparison of this with the year-old children immunised with Td5aP-IVP in the main study indicated a difference of 0.0% [ 2.7 to +5.3%]. There was no significant difference in post-vaccination tetanus or diphtheria GMTs between the two age groups immunised with Td5aP-IPV (Table 2). Responses to other antigens (Table 3) showed no significant difference between the two age groups except for poliomyelitis virus type 2 and pertactin where postvaccination GMTs were somewhat higher in older age group compared to the younger age group (P = 0.02 in each case) Reactogenicity and adverse reactions Local and systemic reactions were similar in the year-old children when compared with the year-old group. There were no serious adverse events and no subjects withdrew due to adverse events. 5. Discussion The primary endpoint of this study was comparison of immunogenicity of a pre-school booster comparing standard and low-dose diphtheria regimens. Post-vaccination titres for the antigens in the study vaccines (Td5aP-IPV, Td5aP) were high in all groups and the proportion exceeding protective thresholds was in the same range as in previous UK studies using standard dose diphtheria-containing vaccines [8,9]. While slightly lower GMTs were observed in younger children this resulted in no diminution of the proportion protected. Baseline immunity to diphtheria was higher at pre-school age in children who had received a single dose of Men C CRM197 containing vaccine as toddlers (aged 1 3 years during 2000) than in the year olds who received three doses of this vaccine as part of an infant primary course administered at the same time as their diphtheria immunisation. Presumably, the explanation for this is that the CRM197 component acted to boost the primary diphtheria course in older children resulting in persistence of higher antibody levels at pre-school age than was achieved by the primary infant course alone. A similar phenomenon was noted by Miller et al. amongst children who had received a CRM197 H. influenzae type b vaccine [9]. Whilst the proportion of children in the study achieving concentrations of diphtheria antibody above recognised seroprotection levels was the same as in previous studies of standard dose diphtheria vaccines, there was a lower diphtheria GMT in those children who received lowdose diphtheria as compared to the standard dose diphtheria group. Although it has been previously demonstrated in a German study that diphtheria antitoxin titres wane over the first year following vaccination, irrespective of the initial titre, the protection afforded by low-dose diphtheria containing combinations may still persist through to adolescence [10]. A serology follow-up on the subjects of our cohort will attempt to assess the kinetics of the antibody response in the context of the UK vaccination schedule.

7 4268 C.L. Collins et al. / Vaccine 22 (2004) The proportion of subjects achieving tetanus antitoxin titres of 0.1 EU/ml were similar in all four groups and all children attained antibody concentrations that were above the level considered to be protective (Table 2). The GMT was highest in the group that received Td5aP + OPV as has been documented in previous adult studies of Td5aP [11] and lowest in the group receiving DT2aP-IPV. Since all vaccines induced antibody titres far higher than the protective threshold, these variations are probably of no clinical significance. Acellular pertussis has recently been added to the preschool booster given to UK children between the ages of 3.5 and 5 years of age in order to combat the rising incidence of pertussis in older children and young adults [12]. Although pertussis has low mortality in these age groups, the circulation of B. pertussis poses significant risk to partially or unimmunised infants. There are no clear serological correlates of protection for pertussis with the efficacy of vaccines being demonstrated in large scale field studies. Because of the uncertainty over serological correlates of protection, it is difficult to interpret the immunogenicity of the pertussis components of the vaccines in our study. The anti-pt titres were higher in the groups of children receiving either Td5aP- IPV or Td5aP + OPV (2.5 g of PT) than in the recipients of DT2aP-IPV (25 g of PT). The anti-fha titres were lower in the Td5aP-IPV group and the Td5aP + OPV group compared with the DT2aP-IPV group but this is probably attributable to the higher dose of FHA in the two-component vaccine. Although there are no agreed antibody concentration thresholds for protection, the efficacy of the five-component acellular pertussis vaccine has been established in two Swedish studies carried out in the 1990s [13,14] and it is noteworthy that the antibody titres to the pertussis antigens in recipients of the five-component vaccines in the present study appear to be similar to those in the Swedish study where protection was demonstrated. In this study, substantially higher polio antibody titres were obtained with IPV than with OPV (Table 3). It is not clear whether this has any implications for protection against polio but since Europe was declared polio free in 2002, it seems prudent for countries in this region to switch to IPV to eliminate the risk of vaccine-associated paralytic poliomyelitis and this study confirms immunogenicity of all vaccines studied. The diphtheria and tetanus components of the pre-school booster are thought to be responsible for the majority of local reactions as has been demonstrated by the similarity in reactogenicity profiles between DT and DTaP vaccines [9]. In our study, all three vaccines were well tolerated with local and systemic reactions of similar magnitude to those reported in other diphtheria combination vaccinations [8].A particular association between diphtheria antibody titres and local reactions was described in adults in the 1940s [15] and it has been suggested that the rates of reactions are higher amongst those with pre-existing immunity. In the 1940s study, individuals with an antibody level >0.01 units/ml experienced more local reactions than the non-immune. However, it is of interest to note that in our study there was no relationship observed between baseline GMT or peak GMT and reactogenicity in each of the study vaccine groups suggesting that diphtheria antibody level may not be an important factor in reactogenicity to vaccines in the pre-school age group. In addition, there was no evidence for higher reactogenicity in the group receiving a higher dose of diphtheria toxoid. Indeed, the incidence of erythema was actually higher in one of the low-dose diphtheria groups, Td5aP + OPV, than in the DT2aP-IPV group. Enhanced immunity to diphtheria in children who have received meningococcal conjugate vaccines containing CRM197 has previously been observed but enhanced reactogenicity to a further dose of CRM197 was not noted in this group [16]. Despite these observations, it remains possible that reactogenicity may be more problematic in countries other than the UK where the pre-school booster is usually the fifth dose of diphtheria that children receive. In conclusion, the vaccines studied, Td5aP-IPV, Td5aP + OPV and DT2aP-IPV are all sufficiently immunogenic to use as a pre-school booster in the UK from as young as 3 years of age, can be administered concomitantly with MMR vaccine and are well tolerated. In spite of the lower diphtheria dose, Td5aP-IPV was not less reactogenic than other vaccines used and it was equivalent in immunogenicity. Acknowledgements We would like to thank the families and General Practitioners in Oxfordshire and Milton Keynes for participating in this study, Shirley Ashmore for administrative support, and Natalie Phillips and Sarah Green for help with sample collection. We are very grateful to Jonathan Van Tam for assistance with the final draft of the manuscript. This trial was undertaken by the chief investigators (AJP and ERM) on behalf of the University of Oxford and sponsored by Aventis Pasteur MSD. AJP has received assistance from Aventis Pasteur MSD for travel to scientific meetings. References [1] Lewis J. The prevention of diphtheria in Canada and Britain, J Social Hist 1986;20: [2] Galazka A. The changing epidemiology of diphtheria in the vaccine era. J Infect Dis 2000;181(Suppl. 1):S2 9. [3] Immunisation against infectious disease. London: HM Stationary Office; [4] Terry L. The association of cases of poliomyelitis with the use of type 3 oral poliomyelitis vaccines. Washington, DC: US Department of Health, Education and Welfare; [5] Galazka AM. The immunological basis for immunization series. Module 2: diphtheria. Geneva: World Health Organisation; 2001 [WHO/EPI/93.12].

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