- Email: [email protected]
Hib and hepatitis B vaccines administered in two simultaneous injections to infants at 2, 4 and 6 months of age
Vaccine 21 (2003) 3593–3600
Comparison of the reactogenicity and immunogenicity of a combined diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated polio (DTPa–HBV–IPV) vaccine, mixed with the Haemophilus influenzae type b (Hib) conjugate vaccine and administered as a single injection, with the DTPa–IPV/Hib and hepatitis B vaccines administered in two simultaneous injections to infants at 2, 4 and 6 months of age J. Ar´ıstegui a , R. Dal-Ré b , J. D´ıez-Delgado c , J. Marés d , J.M. Casanovas e , P. Garc´ıa-Corbeira b,∗ , E. De Frutos f , D. Van Esso g , J. Verdaguer h , J. De la Flor i , F. Moraga j , R. Boceta b , J.A. Garc´ıa-Mart´ınez b b
a Department of Paediatrics, Basurto Hospital, Bilbao, Spain Medical Department, GlaxoSmithKline, PTM, c/Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain c Department of Paediatrics, Torrecárdenas Hospital, Almer´ıa, Spain d Paediatric Institute Marés-Riera, Gerona, Spain e CAP Roquetes, Barcelona, Spain f ABS Canovellas, Barcelona, Spain g ABS Serraparera, Barcelona, Spain h CAP Sant Llàtzer, Barcelona, Spain i CAP Sant Vicenç dels Horts, Barcelona, Spain j Hospital Materno Infantil Vall d’Hebrón, Barcelona, Spain
Received 12 September 2002; received in revised form 26 March 2003; accepted 19 May 2003
Abstract An open, randomised, multicentre trial was performed to compare the reactogenicity and safety profile of the administration of a hexavalent diphtheria-tetanus-acellular pertussis–hepatitis B–inactivated polio (DTPa–HBV–IPV) vaccine administered in one injection mixed with Haemophilus influenzae type b (Hib) conjugate vaccine (Group 1) with that of a pentavalent DTPa–IPV vaccine mixed with a Hib vaccine (DTPa–IPV/Hib), simultaneously administered with HBV (Group 2) in two injections in opposite thighs, as a primary vaccination course, to healthy infants at 2, 4 and 6 months of age. A total of 235 completed the study, 120 from Group 1 and 115 from Group 2. Blood samples (pre-vaccination and 1 month after the third dose) were obtained from a subset of infants (Group 1: 40; Group 2: 31) to assess the immune response to vaccination. Local and general solicited symptoms were recorded by parents on diary cards. Seven hundred and five diary cards (Group 1: 360; Group 2: 345) were collected. The clinically relevant and most commonly reported local reaction was pain (infant cried when the limb was moved) in 2.5% (Group 1) and 1.2% (Group 2) of diary cards. Fever was more frequently reported in Group 1 (21% of diary cards) than in Group 2 (12% of diary cards). However only 3 and 2% of doses in Groups 1 and 2, respectively, were responsible for a rectal temperature between 38.6 and 39.5 ◦ C and only one case (Group 2) had ≥39.5 ◦ C. Other clinically relevant general symptoms were rarely recorded: irritability (2–2.8%), loss of appetite (0.3–0.6%) and drowsiness (0.3–0.3%). All subjects included in the immunogenicity analysis had seroprotective titres to diphtheria, tetanus, polio virus types 1 and 3, Hib. Almost all subjects were seroprotected for anti-polio type 2 and hepatitis B (with the exception of 1 subject in Group 1 for each antigen). The vaccines response rates to pertussis antigens were over 97 and 90% in Groups 1 and 2, respectively. This study shows that, from a clinical perspective, the DTPa–HBV–IPV/Hib vaccine given in a single injection has a similar reactogenicity and safety profile to that of two licensed vaccines (DTPa–IPV/Hib, HBV) given in two simultaneous injections to infants at 2, 4 and 6 months of age. This is a valuable advantage, since in some countries, such as Spain and the UK, an additional injection (for the administration of meningococcal C conjugate vaccine) has been recently included in the infants’ vaccination calendars. © 2003 Elsevier Ltd. All rights reserved.
∗
Corresponding author. Tel.: +34-91-807-0451; fax: +34-91-807-5944. E-mail address: [email protected] (P. Garc´ıa-Corbeira).
0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00420-1
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1. Introduction Delivering a number of antigens in a single injection is regarded as a priority by parents and physicians [1]. This is even more advantageous in countries like the US in which up to 18 or 19 vaccine injections are given to infants over the first 12–15 months of life [2]. However, in some European countries, and elsewhere (e.g. Canada), several combined vaccines have been licensed, most of which are based on diphtheria-tetanus-acellular pertussis (DTPa) vaccine [3]. Thus, infants may receive the DTPa vaccine combined with hepatitis B vaccine (HBV), and/or Haemophilus influenzae type b (Hib) vaccine and/or inactivated polio virus vaccine (IPV) in a single injection. IPV has replaced the oral polio vaccine in a number of countries, for all (four) or for the first two doses [4,5]. It seems reasonable to assume that the DTPa-based combination vaccine that will be best perceived by both health providers and parents would be the vaccine including HBV, Hib and IPV in one injection. The development of this combination was possible after showing that the addition of IPV to the DTPa–HBV/Hib combined vaccines does not impair the development of B. pertussis-antigen specific cell-mediated immune response [6], and that the reduced anti-PRP antibody titres observed when the Hib conjugate vaccine was administered in a single injection with DTPa is not associated with an increase number of invasive Hib cases [7]. In 2000, the European Medicines Evaluation Agency (EMEA, London) licensed two combination vaccines for the prevention of diphtheria, tetanus, polio, invasive Hib diseases, hepatitis B and pertussis. Since the immunogenicity reaction induced by these vaccines should be similar to that observed by their components (already licensed), and the main advantage of these hexavalent vaccines is the possibility of reducing the number of injections per visit to one, it is clear that the reactogenicity and safety profile should be carefully assessed to ensure their future acceptability by the community. This study was conducted to compare the reactogenicity and safety profile of the DTPa–HBV–IPV/Hib vaccine, mixed in one syringe and administered as a single injection, with that of DTPa–IPV/Hib and HBV, administered in two simultaneous injections, to infants as primary vaccination course.
thigh). The study protocol was approved by the Research Ethics Committees (RECs) of the centres involved, and conducted following the Good Clinical Practices guidelines. Written informed consent was obtained from the parents or guardians prior to enrolment. Since meningococcal C conjugate vaccine was included in the vaccination calendars in Spain in September 2000, a modification to the study protocol was approved by RECs to allow the administration of this vaccine to infants in between study vaccination. The primary objective was to compare the incidence of solicited local symptoms over the full vaccination course (three doses) of subjects included in Group 1 with that of subjects randomised to Group 2. The secondary objectives were to compare the incidence of solicited general symptoms, unsolicited symptoms between the two groups over the full vaccination course, and to assess immunogenicity in a subset of subjects (the first 30 infants in each group whose parents agreed to blood sampling) to each of the 10 antigens. Two blood samplings (prior to first dose and 1 month after the third dose) were taken in these infants. The study was conducted between March and December 2000, in full-term healthy infants aged 8–12 weeks at the time of the first vaccination and recruited in nine Spanish centres. The study started some months before obtaining the approval of the GlaxoSmithKline hexavalent combination (InfanrixTM Hexa) by the European Medicines Evaluation Agency. Subjects were eligible for enrolment in the study if they had no previous history study of diphtheria, tetanus, pertussis, hepatitis B, polio and/or Hib vaccination or disease. Infants were excluded from the study if they were participating in other clinical trial, if they had major congenital defects or serious chronic illnesses, had a history of neurological disorders or seizures, had known or suspected immune dysfunction, were born to a mother known to be HIV-positive or HBsAg-positive, had acute disease or rectal temperature ≥38 ◦ C (inmunization deferred), a history of allergic reaction to any of the vaccine components, were undergoing immunosuppresive therapy, had received any immunoglobulin therapy or blood products within 2 months before enrolment or during the trial or had been administered any vaccines or experimental drug or vaccine during 30 days before the start of the trial or during the trial. 2.2. Study vaccines
2. Materials and methods 2.1. Design and subjects This was an open, randomised, multicentre, comparative phase IIIb clinical trial with two groups of healthy infants. Group 1 received the new experimental hexavalent vaccine (DTPa–HBV–IPV/Hib) in one injection on the left anterolateral thigh. Group 2 (control group) received the DTPa–IPV and Hib vaccines mixed in one injection (left anterolateral thigh) and the HBV vaccine in another (right anterolateral
Vaccines were administered as a primary vaccination course at 2, 4 and 6 months of age. Group 1: A 0.5 ml dose of the experimental vaccine (DTPa–HBV–IPV) contained, ≥30 IU diphtheria toxoid, ≥40 IU tetanus toxoid, 25 g of adsorbed pertussis toxoid, 25 g of adsorbed filamentous hemagglutinin, 8 g of adsorbed pertactin, 10 g of recombinant hepatitis B surface antigen, and 40, 8 and 32 D-antigen units of polio virus type 1, 2 and 3, respectively and 0.7 mg aluminum salts (hydroxide and phosphate). The conjugate Hib vaccine was supplied as a lyophilized pellet containing 10 g PRP conjugated to
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20–40 g tetanus toxoid, 0.12 mg of aluminium phosphate and 10 mg of lactose. The mixed administration (DTPa–HBV–IPV/Hib, InfanrixTM Hexa) was prepared by reconstituting the lyophilized Hib (adsorbed) vaccine pellet with liquid DTPa–HBV–IPV vaccine. Group 2: The DTPa–IPV vaccine had the same components as described for DTPa–HBV–IPV, except for the HBV component. The conjugate Hib vaccine was supplied as a lyophilized pellet containing 10 g PRP conjugated to 20–40 g tetanus toxoid and 10 mg of lactose. The mixed administration (DTPa–IPV/Hib, Infanrix–IPV + Hib) was prepared by reconstituting the lyophilized Hib vaccine pellet with the liquid DTPa–IPV vaccine. The HBV vaccine (Engerix-B) contained 10 g of recombinant hepatitis surface B antigen and 0.25 mg of aluminum salts. Vaccines were administered using a needle of 25 G × 5/8 in. at 2 months of age and a needle of 23 G × 1 in. at 4 and 6 months of age to ensure a deep intramuscular injection. All vaccines were manufactured by GlaxoSmithKline Biologicals, former SmithKline Beecham Biologicals (Rixensart, Belgium). Single lots of the vaccines were used. Meningococcal C conjugate vaccine (Meningitec, WyethLederle) was administered with an interval of no less than 30 days after/before the administration of study vaccines in order to be able to evaluate satisfactorily the general reactogenicity and the immunogenicity of the study vaccines. 2.3. Reactogenicity analysis Diary cards were used by parents or guardians to record solicited local reactions (pain, redness and swelling at the injection site) and general symptoms (fever, irritability, loss of appetite, drowsiness or sleeping more than usual) on the day of vaccination and for three subsequent days. Symptoms were graded from 1 to 3 in intensity, total incidence and grade 3 is reported here. Fever was defined as rectal body temperature ≥38.0 ◦ C, and grade 3 as a temperature >39.0 ◦ C. Grade 3 pain was defined as a child crying when limb as moved. Any redness or swelling at the injection site was recorded. A diameter of >20 mm was defined as grade 3. Grade 3 irritability was defined as inconsolable and persistent crying. For all other symptoms, grade 3 was defined as preventing normal daily activities. Unsolicited symptoms during a 30-day follow-up period after each vaccination and serious adverse events during the entire period were also recorded. 2.4. Immunogenicity analysis All serum samples were maintained at −20 ◦ C until serological analyses were performed at GlaxoSmithKline Biologicals (Rixensart, Belgium) in a blinded fashion. Antibodies against diphtheria and tetanus toxoids were measured by enzyme-linked immunosorbent assay (ELISA) techniques, with a 0.1 IU/ml cut-off level [8,9]. Seroprotection was considered to have been attained when titres reached
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≥0.1 mIU/ml. Antibodies (IgG) against PT, FHA and PRN, were measured by ELISA with a cut-off of 5 ELISA units (EL.U)/ml [10]. The assay was calibrated using the pooled US FDA reference sera (lot 3 for PT and FHA; lot 4 for PRN). Vaccine response was defined as the appearance of antibodies in seronegative subjects at pre-vaccination, or at least maintenance of pre-vaccination antibody titres in subjects who were seropositive at pre-vaccination. Anti-HBs antibody titres were determined using a commercially available kit (AUSAB® EIA test, Abbott Laboratories, Chicago, IL) with a 10 mIU/ml cut-off. Titres ≥10 mIU/ml are considered as protective [11]. Total antibodies to Hib polysaccharide polyribosylribitol phosphate (PRP) were measured by ELISA with a 0.15 mcg/ml cut-off [12,13]. Titres ≥0.15 mcg/ml were considered to be seroprotective. Anti-polio virus type 1, 2 and 3 were measured by micro-neutralization test, following (adapted) WHO guidelines, with a 1/8 dilution cut-off (seropositivity: antibody titres ≥8-fold dilution) [14]. 2.5. Sample size and statistical analysis Since a previous study indicated that 66.5% of subjects would experience local symptoms after vaccination with DTPa–HBV–IPV/Hib, the sample size needed to show a difference of at least 15% between groups, would be 118 subjects per group, with a 5% alpha error and 80% power. In order to compensate for a 10% drop-out rate, the targeted sample size was 124 infants per group. A subset of subjects whose parents accepted blood sampling were evaluated for immunogenicity. All infants were evaluated for reactogenicity. Two-sided Fisher’s exact test was used to compare reactogenicity between groups. Significance was assumed at P < 0.05. The Bonferroni method was used to adjust P-values on multiple comparisons [15]. Due to the limited number of cases with blood sampling, no statistical analysis was performed with the immunogenicity data. However, seroprotection/vaccine response rates and Geometric Mean Titres (GMT) were calculated, with a 95% CI for all vaccine components.
3. Results A total of 241 infants (Group 1: 123; Group 2: 118) were enrolled. However, only 235 subjects (Group 1: 120; Group 2: 115) were eligible for the reactogenicity analysis, since one subject (Group 1) did not receive the third dose, three (two from Group 1, one from Group 2) received vaccines forbidden in the protocol, and two (Group 2) were eliminated due to randomisation failure. The immunogenicity subset comprised 71 infants: 40 in Group 1, and 31 in Group 2. There were no statistically significantly differences between groups in relation to mean age and female/male ratio. The mean age (±S.D.) of the study population was
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Table 1 Overall incidence of total and grade 3 local solicited symptoms according to per-dose analysis during a 4-day follow-up period after vaccination Symptom
Grade
Group 1 (N = 359)a DTPa–HBV–IPV/Hib
Group 2 (N = 345)
n
DTPa–IPV/Hib
%
95% CI
Group 2 (N = 345) Any (DTPa–IPV/Hib + HBV)
HBV
n
%
95% CI
n
%
95% CI
n
%
95% CI
Pain
Total Grade 3
81 9
22.6 2.5
18.3–27.2 1.2–4.7
58 4
16.8 1.2
13.0–21.2 0.3–2.9
34 1
9.9 0.3
6.9–13.5 0.0–1.6
61 4
17.7 1.2
13.8–22.1 0.3–2.9
Redness
Total Grade 3
109 4
30.4 1.1
25.6–35.4 0.3–2.8
72 1
20.9 0.3
16.7–25.5 0.0–1.6
40 0
11.6 0.0
8.4–15.5 0.0–1.1
82 1
23.8 0.3
19.4–28.6 0.0–1.6
Swelling
Total Grade 3
81 5
22.6∗ 1.4
18.3–27.2 0.5–3.2
52 3
15.1 0.9
11.5–19.3 0.2–2.5
18 0
5.2 0.0
3.1–8.1 0.0–1.1
57 3
16.5∗ 0.9
12.8–20.9 0.2–2.5
Group 1: DTPa–HBV/Hib (single injection); Group 2: DTPa–HBV + Hib (two injections in opposite limbs); N: total number of diary cards returned following all doses; n: total number of a solicited local symptoms following all doses; total: all specified reactions reported during the follow-up period; grade 3 pain: pain such that the infant cries when limb is moved; grade 3 redness or swelling: greatest diameter >20 mm. a In one subject local symptoms were not completed but diary card was completed for general symptoms. ∗ P < 0.05.
8.7 ± 0.8 weeks old (range: 8–11) and the female/male ratio was 0.77/1, respectively. 3.1. Reactogenicity and safety analysis The numbers of diary cards returned were 360 and 345 for Groups 1 and 2, respectively. Table 1 shows the overall incidence of total and grade 3 solicited local symptoms, according to per-dose analysis, reported during the 4-day follow-up period. When in Group 2 local reaction was counted once (“any” in Table 1), even if it was recorded at both sites (for the separate administration) the incidence (95% CI) of pain, redness and swelling was 17.7% (13.8–22.1), 23.8% (19.4–28.6) and 16.5% (12.8–20.9), respectively; grade 3 figures were 1.2% (0.3–2.9), 0.3% (0.0–1.6) and 0.9% (0.2–2.5), respectively. There were no statistically significant differences between the two groups except for total swelling (P < 0.05).
Table 2 shows the overall and grade 3 incidences of solicited general symptoms according to the per-dose analysis, reported during the 4-day follow-up period. Fever was more commonly reported in Group 1 (21.1%) than in Group 2 (12.2%) (P < 0.05). Only 12 (3.3%) and 7 (2%) doses were followed by a rectal temperature between 38.6 and 39.5 ◦ C in Groups 1 and 2, respectively. One case (Group 2) had grade 3 fever (>39.5 ◦ C). A similar incidence in irritability, loss of appetite and sleepiness was reported for both groups. All grade 3 symptoms (3% of all general symptoms in both groups) were determined by the investigators to be related to the vaccines, and resolved within the 4-day follow-up period. Table 3 shows the incidence of local symptoms and fever per subject at each dose. Of the total 213 unsolicited symptoms (110 in Group 1 and 103 in Group 2), 12 (5.6%) were considered to be related to vaccination (11 and 1 in the mixed and separate groups, respectively). However all resolved uneventfully. Eight of
Table 2 Overall incidence of all and grade 3 solicited general symptoms related to vaccination, according to per-dose analysis, reported during the 4-day follow-up period after vaccination Symptom
Intensity
Fever
≥38 ◦ C
Group 1 (N = 360) DTPa–HBV–IPV/Hib
Group 2 (N = 345) DTPa–IPV/Hib + HBV
n
n
%
95% CI
%
95% CI
>39.5 ◦ C
76 0
21.1∗ 0
17.0–25.7 0.0–1.0
42 1
12.2∗ 0.3
8.9–16.1 0.0–1.6
Irritability
Total Grade 3
143 10
39.7 2.8
34.6–45.0 1.3–5.0
113 7
32.8 2.0
27.8–38.0 0.8–4.1
Loss of appetite
Total Grade 3
91 2
25.3 0.6
20.9–30.1 0.1–2.0
74 1
21.4 0.3
17.2–26.2 0.0–1.6
Drowsiness
Total Grade 3
99 1
27.5 0.3
23.0–32.4 0.0–1.5
100 1
29.0 0.3
24.3–34.1 0.0–1.6
Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of diary cards returned following all doses; n: total number of solicited general symptoms following all doses; total: all reports of a solicited general symptoms; grade 3 irritability: persistent crying that could not be comforted. grade 3 loss of appetite and drowsiness: adverse event that prevented normal, everyday activities. ∗ P < 0.05.
Symptom
Pain Redness Swelling Fever a
Dose 1
Dose 2
Dose 3
Group 1 (N = 120) DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
Group 1 (N = DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
Group 1 (N = 120) DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
25.0 28.3 22.5 14.2
22.6 19.1 13.9 7.8
13.0 11.3 4.3 7.8
21.8 33.6 23.5 33.3
15.7 20.0 13.9 19.1
11.3 13.0 5.2 19.1
20.8 29.2 21.7 15.8
12.2 23.5 17.4 9.6
5.2 10.4 6.1 9.6
119)a
In one subject local symptoms were not completed but diary card was completed for general symptoms.
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Table 3 Incidence (%) of local symptoms and fever per subject at each dose
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Table 4 Seroprotective rates (% SP) and geometric mean titres (GMT) for anti-diphtheria, anti-tetanus and anti-polio virus type 1, 2 and 3 antibodies by group (1 month post-vaccination) Antibody
Anti-diphtheria (≥0.1 IU/ml) Anti-tetanus (≥ 0.1 IU/ml) Anti-polio type 1 (≥8 dilution) Anti-polio type 2 (≥8 dilution) Anti-polio type 3 (≥8 dilution) Anti-HBs (≥10 mIU/ml) Anti-PRP (≥0.15 mcg/ml) Anti-PRP (≥1.0 mcg/ml)
Group 1 (N = 40) DTPa–HBV–IPV/Hib
Group 2 (N = 31) DTPa–IPV/Hib + HBV
n
% SP
95% CI
GMT (95% CI)
n
% SP
95% CI
GMT (95% CI)
40 40 34 31 32 39 40 34
100 100 100 96.9 100 97.5 100 85
91.2–100 91.2–100 89.7–100 83.8–99.9 89.1–100 86.8–99.9 91.2–100 70.2–94.3
3.963 (3.095–5.075) 2.869 (2.114–3.894) 481.6 (322.9–718.3) 350.6 (219.7–559.6) 1152.3 (756.7–1754.5) 970 (573.4–1640.8) 4.662 (3.126–6.951) –
31 31 29 27 28 31 31 29
100 100 100 100 100 100 100 93.5
88.8–100 88.8–100 88.1–100 87.2–100 87.7–100 88.8–100 88.8–100 78.6–99.2
3.498 (2.654–4.611) 3.370 (2.528–4.492) 612.0 (384.9–973.3) 468.1 (274.7–797.7) 850.4 (464.0–1558.6) 1826.8 (1246.4–2677.4) 4.997 (3.132–7.972) –
Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of subject with available results; n: number of subjects with titres ≥ cut-off value (see Section 2). Table 5 Vaccine response rates (% VR) and geometric mean titres (GMT) for anti-PT, anti-FHA and anti-PRN antibodies per group (1 month post-vaccination) Antibody
Anti-PT VR Anti-FHA VR Anti-PRN VR
Group 1 (N = 40) DTPa–HBV–IPV/Hib
Group 2 (N = 31) DTPa–IPV/Hib + HBV
n
% VR
95% CI
GMT (95% CI)
n
% VR
95% CI
GMT (95% CI)
40 40 39
100 100 97.5
91.2–100 91.2–100 86.8–99.9
73.6 (59.3–91.3) 307.3 (241.6–390.8) 177.4 (128.3–245.3)
28 30 30
90.3 96.8 96.8
74.2–98 83.3–99.9 83.3–99.9
52.8 (39.6–70.4) 259.2 (195.4–343.8) 191.5 (142.4–257.6)
VR: appearance of antibodies above the assay cut-off (5 EL.U/ml) in initially seronegative subjects and at least maintenance of pre-vaccination antibody titers in initially seropositive subjects; Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of subject with available results; n: number of subjects showing a VR. PT: pertussis toxin; FHA: filamentous hemagglutinin; PRN: pertactin.
these reports were related to local reactions which extended past the 4-day follow-up period and four were systemic reactions. There were only four serious adverse events reported, which were considered by the investigators to be not related to vaccination. 3.2. Immunogenicity analysis Tables 4 and 5 show seroprotective or vaccine response rates and GMTs for anti-diphtheria, anti-tetanus, anti-polio virus type 1, 2 and 3, anti-HBs, anti-PRP, anti-PT, anti-FHA and anti-PRN 1 month post-vaccination. Hundred percent seroprotection was obtained in both groups for diphtheria, tetanus, polio virus types 1 and 3, Hib. In only two cases, both in Group 1, seroprotective titres were not reached, for anti-polio virus type 2 and hepatitis B. The vaccines response rates to pertussis antigens were over 97 and 90% in Groups 1 and 2, respectively.
4. Discussion HBV, Hib and DTPa vaccines are increasingly used in developed countries as primary vaccination course for infants. IPV is also included in vaccination calendars in several countries for the whole primary vaccination [4,5]. A number of DTPa-based combination vaccines have been developed and are licensed in Europe and Canada [3]. The DTPa–HBV– IPV/Hib vaccine has the largest number of antigens given
in one injection, a fact which should increase acceptance by healthcare providers and parents. This study shows that this hexavalent vaccine given in a single injection has a similar reactogenicity profile to that of two commercially available vaccines (DTPa–IPV/Hib, HBV) given in two simultaneous injections to infants at 2, 4 and 6 months of age. Reactogenicity data are difficult to compare between different studies but the results obtained with the DTPa– HBV–IPV/Hib combination in the present clinical trial are in line with those obtained in previously published studies where the hexavalent combinations was administered at 3, 4 and 5 months schedule [16–18], at a 2–4 months schedule [19] or at the same 2, 4 and 6 months schedule [20]. It should be noted that fever (rectal temperature: ≥38 ◦ C) was more reported in Group 1 (21%) than in Group 2 (12%) in the per-dose analysis. Other studies have found that fever is reported between 11% [17] and 18% [18] of subjects receiving the hexavalent vaccine. In this trial, only one child (Group 2) had fever >39.5 ◦ C; other investigators have reported 0.2–0.4% of cases with high rectal temperature [16,18–20]. Grade 3 incidences of other solicited general symptoms associated with the DTPa–HBV–IPV/Hib vaccine were very rarely reported in this study, i.e. irritability (2.8%), loss of appetite (0.6%) and sleeping more than usual (0.3%), which were similar percentages to those found by Schmitt et al. [18]. The limited number of subjects from whom immunogenicity data are available does not allow any conclusions
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to be drawn. However, the results obtained were similar to those found in previous studies in which several hundreds of infants received the hexavalent vaccine on a 2, 4 and 6 months [21], 3, 4 and 5 months [16] and 2, 3 and 4 months [18] schedule. This study shows, as others have done [16,18,21] that, 1 month after receiving the third DTPa–HBV–IPV/Hib dose, almost all infants were seroprotected/seropositive against the 10 vaccines’ antigens. In this study, anti-HBs GMT after three doses of the hexavalent vaccine were 970 mIU/ml, slightly lower than those found by Blatter et al. (1240 mIU/ml) [21], but better than those reported in other trials (ca. 400 mIU/ml) [16,18]. In terms of seroprotection rates, the response to Hib in this study (100 and 85% of infants showing titres of anti-PRP ≥0.15 and ≥1.0 mcg/ml, respectively) was similar to that reported in other trials (97–100 and 77–84%, respectively) [16,18,21], but better in terms of GMT: 4.66 in this trial versus 2.2–2.65 mcg/ml [16,18,21]. These results are similar or higher than those found with three-dose primary course of four available PRP-conjugate vaccines [22]. However, the anti-PRP GMT has very limited clinical relevance, since PRP-polysaccharide specific B-cell-memory has been demonstrated with various DTPa-based combinations [23–25]. It has been recently shown that in children who received the hexavalent vaccine as primary vaccination, a 20-fold increase of anti-PRP titres was observed 7–11 days after giving a booster of plain PRP [26]. The most important evidence supporting the appropriateness of using DTPa/Hib-based combinations is that in countries, such as Germany, where use of these vaccines has been widespread since 1996, the number of invasive Hib cases is steadily declining [7]. In addition, recent data have shown similar persistence of antibodies for up to 13 months following the administration of DTPa–HBV–IPV/Hib and DTPa–IPV/Hib + HBV to 3-, 4- and 5-month-old infants as primary vaccination [27]. The results obtained in this study show that, from a clinical perspective, the hexavalent (DTPa–HBV–IPV/Hib) vaccine, administered in one injection, has a similar reactogenicity and safety profile to that observed when the DTPa–IPV/ Hib and HBV vaccines are given simultaneously in two injections. The administration in only one injection of 10 antigens would contribute to increasing the acceptability of vaccines targeting infants, in countries where IPV has replaced OPV and in countries (e.g. Spain) in which it is currently being recommended by medical societies [28]. This is even more important in countries (e.g. Spain and the UK), in which other vaccines, such as meningococcal C conjugate vaccine, have been recently included in the infants’ vaccination calendars [29].
Acknowledgements Study supported by a grant from SmithKline Beecham SA, Madrid, Spain.
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References [1] Edwards KM, Decker MD. Challenges for licensure of new diphtheria, tetanus toxoid, acellular pertussis (DTPa) combination vaccines: counterpoint. Pediatr Infect Dis J 1996;15:1070–3. [2] American Academy of Pediatrics. Committee on Infectious Diseases: recommended childood immunization schedule-United Sates. Pediatrics 2000;105:148–51. [3] Decker MD, Edwards KM. Combination vaccines: problems and promise. J Pediatr 2000;137:291–5 [editorial]. [4] Salisbury DM, Dittmann S. Immunization in Europe. In: Plotkin SA, Orenstein WA, editors. Vaccines. 3rd ed. Philadelphia, PA: Saunders; 1999. p. 1033–46. [5] American Academy of Pediatrics. Committee on Infectious Diseases: prevention of poliomyelitis: recommendations for the use of only inactivated poliovirus vaccine for routine immunization. Pediatrics 1999;104:1404–6. [6] Meyer CU, Bilen G, Urschel S, Schuind A, Slauoi M, Clemens R, et al. Integration of IPV into multivalent DTPa–HBV-combination vaccines suggests promotion of pertussis-related T-cellular immune responses. Abstract 698 Presented at the 36th Annual Meeting of the Infectious Diseases Society of America (IDSA). Denver, Colorado; 12–15 November 1998. [7] Schmitt HJ, Von Kries R, Hassenpflug B, Hermann M, Siedler A, Niessing W, et al. Haemophilus influenzae type b disease: impact and effectiveness of diphteria-tetanus toxoids-acellular pertussis (inactivated poliovirus)/H. influenzae type b combination vaccines. Pediatr Infect Dis J 2001;20:767–74. [8] Camargo ME, Silveira L, Furuta JA, Oliveira EPT, Germak OA. Immunoenzymatic assay of anti-diphtheric toxin antibodies in human serum. J Clin Microbiol 1984;20:772–4. [9] Melville-Smith ME, Seagroatt VA, Watkins JT. A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralisation test in mice as a method for the estimation of tetanus antitoxin in human sera. J Biol Stand 1983;11:137–44. [10] Karpinski KF, Hayward S, Tryphonas H. Statistical considerations in the quantitation of serum immunoglobulin levels using the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods 1987;103:189–94. [11] World Health Organization. Progress in the control of viral hepatitis: memorandum for a WHO meeting, Bull WHO 1988;66:443–5. [12] Käythy H, Peltola K, Mäkelä PH. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b. J Infect Dis 1983;147:1100. [13] Anderson P. The protective level of serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b. J Infect Dis 1983;149:1034–5. [14] Standard procedure for determining immunogenicity to poliovirus using microneutralization test (WHO/EPI/GEN 93.9). [15] Altman DG. Practical statistics for medical research. London: Chapman and Hall; 1991. [16] Zepp F, Schuind A, Habermehl P, Huff J, Jahn K, Thoelen S. Evolution of combination vaccines: comparison of two candidate vaccines with licensed vaccines. Abstract P30 Presented at 18th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID). Noordwijk, The Netherlands; 3–5 May 2000. [17] Jahn K, Huff J, Schund A, Kowalzik F, Faber J, Habermehl P, et al. Multi-center primary vaccination trial to assess the safety of a hexavalent DTPa–HBV–IPV/Hib vaccine in infants. Abstract 58 Presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Toronto, Canada; 17–20 September 2000. [18] Schmitt HJ, Knuf M, Ortiz E, Sanger R, Uwamwezi MC, Kaufhold A. Primary vaccination of infants with diphtheria-tetanus-acellular pertussis–hepatitis B virus–inactivated polio virus and Haemophilus influenzae type b vaccines given as either separate or mixed injections. J Pediatr 2000;137:304–12.
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[19] Cohen R, Schuerman L. Reactogenicity of a new DTPa–HBV–IPV (+ &/Hib) vaccines after primary and booster doses. Abstract P31 Presented at 18th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID). Noordwijk, The Netherlands; 3–5 May 2000. [20] Blatter M, Pichichero M, Harrison C, Johnson C, Steinhoff M, Senders S, et al. A combination DTPa–HepB–IPV/Hib vaccine administered to infants at 2, 4 and 6 months of age with or without a hepatitis B vaccine birth dose. Abstract 626 Presented at the 38th Annual Meeting of the Infectious Diseases Society of America (IDSA). New Orleans, Louisiana; 7–10 September 2000. [21] Blatter MM, Reisinger KS, Terwelp DR, DelBuono FJ, Howe BJ. Immunogenicity of a combined diphtheria-tetanus-acellular pertussis (DT-tricomponent Pa)–hepatitis B (HB)–inactivated polioviru (IPV) admixed with Haemophilus influenzae type b (Hib) vaccine in infants. Abstract 813 Presented at The American Pediatric Society and The Society for Pediatric Research Meeting. New Orleans, Louisiana; 1–5 May 1998. Pediatr Res 1998;43:141A. [22] Decker MD, Edwards KM, Bradley R, Palmer P. Comparative trial in infants of four conjugate Haemophilus influenzae type b vaccines. J Pediatr 1992;120:184–9. [23] Zepp F, Scmitt HJ, Kaufhold A, Scuind A, Knuf M, Habermehl P, et al. Evidence for induction of polysaccharide specific B-cell-memory in the 1st year of life: plain Haemophilus influenzae type b PRP (Hib) boosters in children primed with tetanus-conjugate Hib–DTPa–HBV combined vaccine. Eur J Pediatr 1997;156:18–24. [24] Schmitt HJ, Zepp F, Muschenborn S, Sumenicht C, Schuind A, Beutel K, et al. Immunogenicity and reactogenicity of a Haemophilus influenzae type b tetanus conjugate vaccine when administered
[25]
[26]
[27]
[28]
[29]
separately or mixed with concomitant diphtheria-tetanus-toxoid and acellular pertussis vaccine for primary and for booster immunizations. Eur J Pediatr 1998;157:208–14. Zepp F, Meyer CU, Kowalzol P, Habermehl P, Stahmer S, Ortiz E, et al. Evaluation of the immunological memory induced by primary vaccination with Haemophilus influenzae type b tetanus conjugate vaccine co-administered with various DTPa-based vaccines. Abstract 242 Presented at the 439th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). San Francisco, California; 26–29 September 1999. Eskola J, Ward J, Dagan R, Goldblatt D, Zepp F, Siegrist CA. Combined vaccination of Haemophilus influenzae type b conjugate and diphteria-tetanus-pertussis containing acellular pertussis. Lancet 1999;354:2063–8. Schmitt HJ, Knuf M, Beutel K, Schuerman L. Antibody persistence, PRP specific immune memory and booster responses in children primed with a new combination vaccine DTPa–HBV–IPV given as either a mixed or separate injection. Abstract P29 Presented at 18th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID). Noordwijk, The Netherlands; 3–5 May 2000. Heininger U, Schuerman L. Evidence for pre-booster antibody persistence in an open randomized study on infants primed with DTPa–HBV–IPV/Hib or DTPa–IPV/Hib + HBV vaccines at 3–5 months of age. Abstract Presented at 19th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID). Istambul, Turkey; 26–28 March 2001. Comité Asesor de Vacunas de la AEP. Calendario vacunal de la Asociación Española de Pediatr´ıa 2001–2002. An Esp Pediatr 2001;55:30–8.
Comparison of the reactogenicity and immunogenicity of a combined diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated polio (DTPa–HBV–IPV) vaccine, mixed with the Haemophilus influenzae type b (Hib) conjugate vaccine and administered as a single injection, with the DTPa–IPV/Hib and hepatitis B vaccines administered in two simultaneous injections to infants at 2, 4 and 6 months of age J. Ar´ıstegui a , R. Dal-Ré b , J. D´ıez-Delgado c , J. Marés d , J.M. Casanovas e , P. Garc´ıa-Corbeira b,∗ , E. De Frutos f , D. Van Esso g , J. Verdaguer h , J. De la Flor i , F. Moraga j , R. Boceta b , J.A. Garc´ıa-Mart´ınez b b
a Department of Paediatrics, Basurto Hospital, Bilbao, Spain Medical Department, GlaxoSmithKline, PTM, c/Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain c Department of Paediatrics, Torrecárdenas Hospital, Almer´ıa, Spain d Paediatric Institute Marés-Riera, Gerona, Spain e CAP Roquetes, Barcelona, Spain f ABS Canovellas, Barcelona, Spain g ABS Serraparera, Barcelona, Spain h CAP Sant Llàtzer, Barcelona, Spain i CAP Sant Vicenç dels Horts, Barcelona, Spain j Hospital Materno Infantil Vall d’Hebrón, Barcelona, Spain
Received 12 September 2002; received in revised form 26 March 2003; accepted 19 May 2003
Abstract An open, randomised, multicentre trial was performed to compare the reactogenicity and safety profile of the administration of a hexavalent diphtheria-tetanus-acellular pertussis–hepatitis B–inactivated polio (DTPa–HBV–IPV) vaccine administered in one injection mixed with Haemophilus influenzae type b (Hib) conjugate vaccine (Group 1) with that of a pentavalent DTPa–IPV vaccine mixed with a Hib vaccine (DTPa–IPV/Hib), simultaneously administered with HBV (Group 2) in two injections in opposite thighs, as a primary vaccination course, to healthy infants at 2, 4 and 6 months of age. A total of 235 completed the study, 120 from Group 1 and 115 from Group 2. Blood samples (pre-vaccination and 1 month after the third dose) were obtained from a subset of infants (Group 1: 40; Group 2: 31) to assess the immune response to vaccination. Local and general solicited symptoms were recorded by parents on diary cards. Seven hundred and five diary cards (Group 1: 360; Group 2: 345) were collected. The clinically relevant and most commonly reported local reaction was pain (infant cried when the limb was moved) in 2.5% (Group 1) and 1.2% (Group 2) of diary cards. Fever was more frequently reported in Group 1 (21% of diary cards) than in Group 2 (12% of diary cards). However only 3 and 2% of doses in Groups 1 and 2, respectively, were responsible for a rectal temperature between 38.6 and 39.5 ◦ C and only one case (Group 2) had ≥39.5 ◦ C. Other clinically relevant general symptoms were rarely recorded: irritability (2–2.8%), loss of appetite (0.3–0.6%) and drowsiness (0.3–0.3%). All subjects included in the immunogenicity analysis had seroprotective titres to diphtheria, tetanus, polio virus types 1 and 3, Hib. Almost all subjects were seroprotected for anti-polio type 2 and hepatitis B (with the exception of 1 subject in Group 1 for each antigen). The vaccines response rates to pertussis antigens were over 97 and 90% in Groups 1 and 2, respectively. This study shows that, from a clinical perspective, the DTPa–HBV–IPV/Hib vaccine given in a single injection has a similar reactogenicity and safety profile to that of two licensed vaccines (DTPa–IPV/Hib, HBV) given in two simultaneous injections to infants at 2, 4 and 6 months of age. This is a valuable advantage, since in some countries, such as Spain and the UK, an additional injection (for the administration of meningococcal C conjugate vaccine) has been recently included in the infants’ vaccination calendars. © 2003 Elsevier Ltd. All rights reserved.
∗
Corresponding author. Tel.: +34-91-807-0451; fax: +34-91-807-5944. E-mail address: [email protected] (P. Garc´ıa-Corbeira).
0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00420-1
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1. Introduction Delivering a number of antigens in a single injection is regarded as a priority by parents and physicians [1]. This is even more advantageous in countries like the US in which up to 18 or 19 vaccine injections are given to infants over the first 12–15 months of life [2]. However, in some European countries, and elsewhere (e.g. Canada), several combined vaccines have been licensed, most of which are based on diphtheria-tetanus-acellular pertussis (DTPa) vaccine [3]. Thus, infants may receive the DTPa vaccine combined with hepatitis B vaccine (HBV), and/or Haemophilus influenzae type b (Hib) vaccine and/or inactivated polio virus vaccine (IPV) in a single injection. IPV has replaced the oral polio vaccine in a number of countries, for all (four) or for the first two doses [4,5]. It seems reasonable to assume that the DTPa-based combination vaccine that will be best perceived by both health providers and parents would be the vaccine including HBV, Hib and IPV in one injection. The development of this combination was possible after showing that the addition of IPV to the DTPa–HBV/Hib combined vaccines does not impair the development of B. pertussis-antigen specific cell-mediated immune response [6], and that the reduced anti-PRP antibody titres observed when the Hib conjugate vaccine was administered in a single injection with DTPa is not associated with an increase number of invasive Hib cases [7]. In 2000, the European Medicines Evaluation Agency (EMEA, London) licensed two combination vaccines for the prevention of diphtheria, tetanus, polio, invasive Hib diseases, hepatitis B and pertussis. Since the immunogenicity reaction induced by these vaccines should be similar to that observed by their components (already licensed), and the main advantage of these hexavalent vaccines is the possibility of reducing the number of injections per visit to one, it is clear that the reactogenicity and safety profile should be carefully assessed to ensure their future acceptability by the community. This study was conducted to compare the reactogenicity and safety profile of the DTPa–HBV–IPV/Hib vaccine, mixed in one syringe and administered as a single injection, with that of DTPa–IPV/Hib and HBV, administered in two simultaneous injections, to infants as primary vaccination course.
thigh). The study protocol was approved by the Research Ethics Committees (RECs) of the centres involved, and conducted following the Good Clinical Practices guidelines. Written informed consent was obtained from the parents or guardians prior to enrolment. Since meningococcal C conjugate vaccine was included in the vaccination calendars in Spain in September 2000, a modification to the study protocol was approved by RECs to allow the administration of this vaccine to infants in between study vaccination. The primary objective was to compare the incidence of solicited local symptoms over the full vaccination course (three doses) of subjects included in Group 1 with that of subjects randomised to Group 2. The secondary objectives were to compare the incidence of solicited general symptoms, unsolicited symptoms between the two groups over the full vaccination course, and to assess immunogenicity in a subset of subjects (the first 30 infants in each group whose parents agreed to blood sampling) to each of the 10 antigens. Two blood samplings (prior to first dose and 1 month after the third dose) were taken in these infants. The study was conducted between March and December 2000, in full-term healthy infants aged 8–12 weeks at the time of the first vaccination and recruited in nine Spanish centres. The study started some months before obtaining the approval of the GlaxoSmithKline hexavalent combination (InfanrixTM Hexa) by the European Medicines Evaluation Agency. Subjects were eligible for enrolment in the study if they had no previous history study of diphtheria, tetanus, pertussis, hepatitis B, polio and/or Hib vaccination or disease. Infants were excluded from the study if they were participating in other clinical trial, if they had major congenital defects or serious chronic illnesses, had a history of neurological disorders or seizures, had known or suspected immune dysfunction, were born to a mother known to be HIV-positive or HBsAg-positive, had acute disease or rectal temperature ≥38 ◦ C (inmunization deferred), a history of allergic reaction to any of the vaccine components, were undergoing immunosuppresive therapy, had received any immunoglobulin therapy or blood products within 2 months before enrolment or during the trial or had been administered any vaccines or experimental drug or vaccine during 30 days before the start of the trial or during the trial. 2.2. Study vaccines
2. Materials and methods 2.1. Design and subjects This was an open, randomised, multicentre, comparative phase IIIb clinical trial with two groups of healthy infants. Group 1 received the new experimental hexavalent vaccine (DTPa–HBV–IPV/Hib) in one injection on the left anterolateral thigh. Group 2 (control group) received the DTPa–IPV and Hib vaccines mixed in one injection (left anterolateral thigh) and the HBV vaccine in another (right anterolateral
Vaccines were administered as a primary vaccination course at 2, 4 and 6 months of age. Group 1: A 0.5 ml dose of the experimental vaccine (DTPa–HBV–IPV) contained, ≥30 IU diphtheria toxoid, ≥40 IU tetanus toxoid, 25 g of adsorbed pertussis toxoid, 25 g of adsorbed filamentous hemagglutinin, 8 g of adsorbed pertactin, 10 g of recombinant hepatitis B surface antigen, and 40, 8 and 32 D-antigen units of polio virus type 1, 2 and 3, respectively and 0.7 mg aluminum salts (hydroxide and phosphate). The conjugate Hib vaccine was supplied as a lyophilized pellet containing 10 g PRP conjugated to
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20–40 g tetanus toxoid, 0.12 mg of aluminium phosphate and 10 mg of lactose. The mixed administration (DTPa–HBV–IPV/Hib, InfanrixTM Hexa) was prepared by reconstituting the lyophilized Hib (adsorbed) vaccine pellet with liquid DTPa–HBV–IPV vaccine. Group 2: The DTPa–IPV vaccine had the same components as described for DTPa–HBV–IPV, except for the HBV component. The conjugate Hib vaccine was supplied as a lyophilized pellet containing 10 g PRP conjugated to 20–40 g tetanus toxoid and 10 mg of lactose. The mixed administration (DTPa–IPV/Hib, Infanrix–IPV + Hib) was prepared by reconstituting the lyophilized Hib vaccine pellet with the liquid DTPa–IPV vaccine. The HBV vaccine (Engerix-B) contained 10 g of recombinant hepatitis surface B antigen and 0.25 mg of aluminum salts. Vaccines were administered using a needle of 25 G × 5/8 in. at 2 months of age and a needle of 23 G × 1 in. at 4 and 6 months of age to ensure a deep intramuscular injection. All vaccines were manufactured by GlaxoSmithKline Biologicals, former SmithKline Beecham Biologicals (Rixensart, Belgium). Single lots of the vaccines were used. Meningococcal C conjugate vaccine (Meningitec, WyethLederle) was administered with an interval of no less than 30 days after/before the administration of study vaccines in order to be able to evaluate satisfactorily the general reactogenicity and the immunogenicity of the study vaccines. 2.3. Reactogenicity analysis Diary cards were used by parents or guardians to record solicited local reactions (pain, redness and swelling at the injection site) and general symptoms (fever, irritability, loss of appetite, drowsiness or sleeping more than usual) on the day of vaccination and for three subsequent days. Symptoms were graded from 1 to 3 in intensity, total incidence and grade 3 is reported here. Fever was defined as rectal body temperature ≥38.0 ◦ C, and grade 3 as a temperature >39.0 ◦ C. Grade 3 pain was defined as a child crying when limb as moved. Any redness or swelling at the injection site was recorded. A diameter of >20 mm was defined as grade 3. Grade 3 irritability was defined as inconsolable and persistent crying. For all other symptoms, grade 3 was defined as preventing normal daily activities. Unsolicited symptoms during a 30-day follow-up period after each vaccination and serious adverse events during the entire period were also recorded. 2.4. Immunogenicity analysis All serum samples were maintained at −20 ◦ C until serological analyses were performed at GlaxoSmithKline Biologicals (Rixensart, Belgium) in a blinded fashion. Antibodies against diphtheria and tetanus toxoids were measured by enzyme-linked immunosorbent assay (ELISA) techniques, with a 0.1 IU/ml cut-off level [8,9]. Seroprotection was considered to have been attained when titres reached
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≥0.1 mIU/ml. Antibodies (IgG) against PT, FHA and PRN, were measured by ELISA with a cut-off of 5 ELISA units (EL.U)/ml [10]. The assay was calibrated using the pooled US FDA reference sera (lot 3 for PT and FHA; lot 4 for PRN). Vaccine response was defined as the appearance of antibodies in seronegative subjects at pre-vaccination, or at least maintenance of pre-vaccination antibody titres in subjects who were seropositive at pre-vaccination. Anti-HBs antibody titres were determined using a commercially available kit (AUSAB® EIA test, Abbott Laboratories, Chicago, IL) with a 10 mIU/ml cut-off. Titres ≥10 mIU/ml are considered as protective [11]. Total antibodies to Hib polysaccharide polyribosylribitol phosphate (PRP) were measured by ELISA with a 0.15 mcg/ml cut-off [12,13]. Titres ≥0.15 mcg/ml were considered to be seroprotective. Anti-polio virus type 1, 2 and 3 were measured by micro-neutralization test, following (adapted) WHO guidelines, with a 1/8 dilution cut-off (seropositivity: antibody titres ≥8-fold dilution) [14]. 2.5. Sample size and statistical analysis Since a previous study indicated that 66.5% of subjects would experience local symptoms after vaccination with DTPa–HBV–IPV/Hib, the sample size needed to show a difference of at least 15% between groups, would be 118 subjects per group, with a 5% alpha error and 80% power. In order to compensate for a 10% drop-out rate, the targeted sample size was 124 infants per group. A subset of subjects whose parents accepted blood sampling were evaluated for immunogenicity. All infants were evaluated for reactogenicity. Two-sided Fisher’s exact test was used to compare reactogenicity between groups. Significance was assumed at P < 0.05. The Bonferroni method was used to adjust P-values on multiple comparisons [15]. Due to the limited number of cases with blood sampling, no statistical analysis was performed with the immunogenicity data. However, seroprotection/vaccine response rates and Geometric Mean Titres (GMT) were calculated, with a 95% CI for all vaccine components.
3. Results A total of 241 infants (Group 1: 123; Group 2: 118) were enrolled. However, only 235 subjects (Group 1: 120; Group 2: 115) were eligible for the reactogenicity analysis, since one subject (Group 1) did not receive the third dose, three (two from Group 1, one from Group 2) received vaccines forbidden in the protocol, and two (Group 2) were eliminated due to randomisation failure. The immunogenicity subset comprised 71 infants: 40 in Group 1, and 31 in Group 2. There were no statistically significantly differences between groups in relation to mean age and female/male ratio. The mean age (±S.D.) of the study population was
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Table 1 Overall incidence of total and grade 3 local solicited symptoms according to per-dose analysis during a 4-day follow-up period after vaccination Symptom
Grade
Group 1 (N = 359)a DTPa–HBV–IPV/Hib
Group 2 (N = 345)
n
DTPa–IPV/Hib
%
95% CI
Group 2 (N = 345) Any (DTPa–IPV/Hib + HBV)
HBV
n
%
95% CI
n
%
95% CI
n
%
95% CI
Pain
Total Grade 3
81 9
22.6 2.5
18.3–27.2 1.2–4.7
58 4
16.8 1.2
13.0–21.2 0.3–2.9
34 1
9.9 0.3
6.9–13.5 0.0–1.6
61 4
17.7 1.2
13.8–22.1 0.3–2.9
Redness
Total Grade 3
109 4
30.4 1.1
25.6–35.4 0.3–2.8
72 1
20.9 0.3
16.7–25.5 0.0–1.6
40 0
11.6 0.0
8.4–15.5 0.0–1.1
82 1
23.8 0.3
19.4–28.6 0.0–1.6
Swelling
Total Grade 3
81 5
22.6∗ 1.4
18.3–27.2 0.5–3.2
52 3
15.1 0.9
11.5–19.3 0.2–2.5
18 0
5.2 0.0
3.1–8.1 0.0–1.1
57 3
16.5∗ 0.9
12.8–20.9 0.2–2.5
Group 1: DTPa–HBV/Hib (single injection); Group 2: DTPa–HBV + Hib (two injections in opposite limbs); N: total number of diary cards returned following all doses; n: total number of a solicited local symptoms following all doses; total: all specified reactions reported during the follow-up period; grade 3 pain: pain such that the infant cries when limb is moved; grade 3 redness or swelling: greatest diameter >20 mm. a In one subject local symptoms were not completed but diary card was completed for general symptoms. ∗ P < 0.05.
8.7 ± 0.8 weeks old (range: 8–11) and the female/male ratio was 0.77/1, respectively. 3.1. Reactogenicity and safety analysis The numbers of diary cards returned were 360 and 345 for Groups 1 and 2, respectively. Table 1 shows the overall incidence of total and grade 3 solicited local symptoms, according to per-dose analysis, reported during the 4-day follow-up period. When in Group 2 local reaction was counted once (“any” in Table 1), even if it was recorded at both sites (for the separate administration) the incidence (95% CI) of pain, redness and swelling was 17.7% (13.8–22.1), 23.8% (19.4–28.6) and 16.5% (12.8–20.9), respectively; grade 3 figures were 1.2% (0.3–2.9), 0.3% (0.0–1.6) and 0.9% (0.2–2.5), respectively. There were no statistically significant differences between the two groups except for total swelling (P < 0.05).
Table 2 shows the overall and grade 3 incidences of solicited general symptoms according to the per-dose analysis, reported during the 4-day follow-up period. Fever was more commonly reported in Group 1 (21.1%) than in Group 2 (12.2%) (P < 0.05). Only 12 (3.3%) and 7 (2%) doses were followed by a rectal temperature between 38.6 and 39.5 ◦ C in Groups 1 and 2, respectively. One case (Group 2) had grade 3 fever (>39.5 ◦ C). A similar incidence in irritability, loss of appetite and sleepiness was reported for both groups. All grade 3 symptoms (3% of all general symptoms in both groups) were determined by the investigators to be related to the vaccines, and resolved within the 4-day follow-up period. Table 3 shows the incidence of local symptoms and fever per subject at each dose. Of the total 213 unsolicited symptoms (110 in Group 1 and 103 in Group 2), 12 (5.6%) were considered to be related to vaccination (11 and 1 in the mixed and separate groups, respectively). However all resolved uneventfully. Eight of
Table 2 Overall incidence of all and grade 3 solicited general symptoms related to vaccination, according to per-dose analysis, reported during the 4-day follow-up period after vaccination Symptom
Intensity
Fever
≥38 ◦ C
Group 1 (N = 360) DTPa–HBV–IPV/Hib
Group 2 (N = 345) DTPa–IPV/Hib + HBV
n
n
%
95% CI
%
95% CI
>39.5 ◦ C
76 0
21.1∗ 0
17.0–25.7 0.0–1.0
42 1
12.2∗ 0.3
8.9–16.1 0.0–1.6
Irritability
Total Grade 3
143 10
39.7 2.8
34.6–45.0 1.3–5.0
113 7
32.8 2.0
27.8–38.0 0.8–4.1
Loss of appetite
Total Grade 3
91 2
25.3 0.6
20.9–30.1 0.1–2.0
74 1
21.4 0.3
17.2–26.2 0.0–1.6
Drowsiness
Total Grade 3
99 1
27.5 0.3
23.0–32.4 0.0–1.5
100 1
29.0 0.3
24.3–34.1 0.0–1.6
Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of diary cards returned following all doses; n: total number of solicited general symptoms following all doses; total: all reports of a solicited general symptoms; grade 3 irritability: persistent crying that could not be comforted. grade 3 loss of appetite and drowsiness: adverse event that prevented normal, everyday activities. ∗ P < 0.05.
Symptom
Pain Redness Swelling Fever a
Dose 1
Dose 2
Dose 3
Group 1 (N = 120) DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
Group 1 (N = DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
Group 1 (N = 120) DTPa–HBV–IPV/Hib
Group 2 (N = 115) DTPa–IPV/Hib
Group 2 (N = 115) HBV
25.0 28.3 22.5 14.2
22.6 19.1 13.9 7.8
13.0 11.3 4.3 7.8
21.8 33.6 23.5 33.3
15.7 20.0 13.9 19.1
11.3 13.0 5.2 19.1
20.8 29.2 21.7 15.8
12.2 23.5 17.4 9.6
5.2 10.4 6.1 9.6
119)a
In one subject local symptoms were not completed but diary card was completed for general symptoms.
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Table 3 Incidence (%) of local symptoms and fever per subject at each dose
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Table 4 Seroprotective rates (% SP) and geometric mean titres (GMT) for anti-diphtheria, anti-tetanus and anti-polio virus type 1, 2 and 3 antibodies by group (1 month post-vaccination) Antibody
Anti-diphtheria (≥0.1 IU/ml) Anti-tetanus (≥ 0.1 IU/ml) Anti-polio type 1 (≥8 dilution) Anti-polio type 2 (≥8 dilution) Anti-polio type 3 (≥8 dilution) Anti-HBs (≥10 mIU/ml) Anti-PRP (≥0.15 mcg/ml) Anti-PRP (≥1.0 mcg/ml)
Group 1 (N = 40) DTPa–HBV–IPV/Hib
Group 2 (N = 31) DTPa–IPV/Hib + HBV
n
% SP
95% CI
GMT (95% CI)
n
% SP
95% CI
GMT (95% CI)
40 40 34 31 32 39 40 34
100 100 100 96.9 100 97.5 100 85
91.2–100 91.2–100 89.7–100 83.8–99.9 89.1–100 86.8–99.9 91.2–100 70.2–94.3
3.963 (3.095–5.075) 2.869 (2.114–3.894) 481.6 (322.9–718.3) 350.6 (219.7–559.6) 1152.3 (756.7–1754.5) 970 (573.4–1640.8) 4.662 (3.126–6.951) –
31 31 29 27 28 31 31 29
100 100 100 100 100 100 100 93.5
88.8–100 88.8–100 88.1–100 87.2–100 87.7–100 88.8–100 88.8–100 78.6–99.2
3.498 (2.654–4.611) 3.370 (2.528–4.492) 612.0 (384.9–973.3) 468.1 (274.7–797.7) 850.4 (464.0–1558.6) 1826.8 (1246.4–2677.4) 4.997 (3.132–7.972) –
Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of subject with available results; n: number of subjects with titres ≥ cut-off value (see Section 2). Table 5 Vaccine response rates (% VR) and geometric mean titres (GMT) for anti-PT, anti-FHA and anti-PRN antibodies per group (1 month post-vaccination) Antibody
Anti-PT VR Anti-FHA VR Anti-PRN VR
Group 1 (N = 40) DTPa–HBV–IPV/Hib
Group 2 (N = 31) DTPa–IPV/Hib + HBV
n
% VR
95% CI
GMT (95% CI)
n
% VR
95% CI
GMT (95% CI)
40 40 39
100 100 97.5
91.2–100 91.2–100 86.8–99.9
73.6 (59.3–91.3) 307.3 (241.6–390.8) 177.4 (128.3–245.3)
28 30 30
90.3 96.8 96.8
74.2–98 83.3–99.9 83.3–99.9
52.8 (39.6–70.4) 259.2 (195.4–343.8) 191.5 (142.4–257.6)
VR: appearance of antibodies above the assay cut-off (5 EL.U/ml) in initially seronegative subjects and at least maintenance of pre-vaccination antibody titers in initially seropositive subjects; Group 1: DTPa–HBV–IPV/Hib (single injection); Group 2: DTPa–IPV/Hib + HBV (two injections in opposite limbs); N: total number of subject with available results; n: number of subjects showing a VR. PT: pertussis toxin; FHA: filamentous hemagglutinin; PRN: pertactin.
these reports were related to local reactions which extended past the 4-day follow-up period and four were systemic reactions. There were only four serious adverse events reported, which were considered by the investigators to be not related to vaccination. 3.2. Immunogenicity analysis Tables 4 and 5 show seroprotective or vaccine response rates and GMTs for anti-diphtheria, anti-tetanus, anti-polio virus type 1, 2 and 3, anti-HBs, anti-PRP, anti-PT, anti-FHA and anti-PRN 1 month post-vaccination. Hundred percent seroprotection was obtained in both groups for diphtheria, tetanus, polio virus types 1 and 3, Hib. In only two cases, both in Group 1, seroprotective titres were not reached, for anti-polio virus type 2 and hepatitis B. The vaccines response rates to pertussis antigens were over 97 and 90% in Groups 1 and 2, respectively.
4. Discussion HBV, Hib and DTPa vaccines are increasingly used in developed countries as primary vaccination course for infants. IPV is also included in vaccination calendars in several countries for the whole primary vaccination [4,5]. A number of DTPa-based combination vaccines have been developed and are licensed in Europe and Canada [3]. The DTPa–HBV– IPV/Hib vaccine has the largest number of antigens given
in one injection, a fact which should increase acceptance by healthcare providers and parents. This study shows that this hexavalent vaccine given in a single injection has a similar reactogenicity profile to that of two commercially available vaccines (DTPa–IPV/Hib, HBV) given in two simultaneous injections to infants at 2, 4 and 6 months of age. Reactogenicity data are difficult to compare between different studies but the results obtained with the DTPa– HBV–IPV/Hib combination in the present clinical trial are in line with those obtained in previously published studies where the hexavalent combinations was administered at 3, 4 and 5 months schedule [16–18], at a 2–4 months schedule [19] or at the same 2, 4 and 6 months schedule [20]. It should be noted that fever (rectal temperature: ≥38 ◦ C) was more reported in Group 1 (21%) than in Group 2 (12%) in the per-dose analysis. Other studies have found that fever is reported between 11% [17] and 18% [18] of subjects receiving the hexavalent vaccine. In this trial, only one child (Group 2) had fever >39.5 ◦ C; other investigators have reported 0.2–0.4% of cases with high rectal temperature [16,18–20]. Grade 3 incidences of other solicited general symptoms associated with the DTPa–HBV–IPV/Hib vaccine were very rarely reported in this study, i.e. irritability (2.8%), loss of appetite (0.6%) and sleeping more than usual (0.3%), which were similar percentages to those found by Schmitt et al. [18]. The limited number of subjects from whom immunogenicity data are available does not allow any conclusions
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to be drawn. However, the results obtained were similar to those found in previous studies in which several hundreds of infants received the hexavalent vaccine on a 2, 4 and 6 months [21], 3, 4 and 5 months [16] and 2, 3 and 4 months [18] schedule. This study shows, as others have done [16,18,21] that, 1 month after receiving the third DTPa–HBV–IPV/Hib dose, almost all infants were seroprotected/seropositive against the 10 vaccines’ antigens. In this study, anti-HBs GMT after three doses of the hexavalent vaccine were 970 mIU/ml, slightly lower than those found by Blatter et al. (1240 mIU/ml) [21], but better than those reported in other trials (ca. 400 mIU/ml) [16,18]. In terms of seroprotection rates, the response to Hib in this study (100 and 85% of infants showing titres of anti-PRP ≥0.15 and ≥1.0 mcg/ml, respectively) was similar to that reported in other trials (97–100 and 77–84%, respectively) [16,18,21], but better in terms of GMT: 4.66 in this trial versus 2.2–2.65 mcg/ml [16,18,21]. These results are similar or higher than those found with three-dose primary course of four available PRP-conjugate vaccines [22]. However, the anti-PRP GMT has very limited clinical relevance, since PRP-polysaccharide specific B-cell-memory has been demonstrated with various DTPa-based combinations [23–25]. It has been recently shown that in children who received the hexavalent vaccine as primary vaccination, a 20-fold increase of anti-PRP titres was observed 7–11 days after giving a booster of plain PRP [26]. The most important evidence supporting the appropriateness of using DTPa/Hib-based combinations is that in countries, such as Germany, where use of these vaccines has been widespread since 1996, the number of invasive Hib cases is steadily declining [7]. In addition, recent data have shown similar persistence of antibodies for up to 13 months following the administration of DTPa–HBV–IPV/Hib and DTPa–IPV/Hib + HBV to 3-, 4- and 5-month-old infants as primary vaccination [27]. The results obtained in this study show that, from a clinical perspective, the hexavalent (DTPa–HBV–IPV/Hib) vaccine, administered in one injection, has a similar reactogenicity and safety profile to that observed when the DTPa–IPV/ Hib and HBV vaccines are given simultaneously in two injections. The administration in only one injection of 10 antigens would contribute to increasing the acceptability of vaccines targeting infants, in countries where IPV has replaced OPV and in countries (e.g. Spain) in which it is currently being recommended by medical societies [28]. This is even more important in countries (e.g. Spain and the UK), in which other vaccines, such as meningococcal C conjugate vaccine, have been recently included in the infants’ vaccination calendars [29].
Acknowledgements Study supported by a grant from SmithKline Beecham SA, Madrid, Spain.
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