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Immunogenicity and reactogenicity of diphtheria, tetanus and pertussis toxoids combined with inactivated polio vaccine, when administered concomitantly with or as a diluent for a Hib conjugate vaccine
Vaccine 19 (2001) 4396– 4403 www.elsevier.com/locate/vaccine
Immunogenicity and reactogenicity of diphtheria, tetanus and pertussis toxoids combined with inactivated polio vaccine, when administered concomitantly with or as a diluent for a Hib conjugate vaccine Nina Knutsson a,*, Birger Trollfors a, John Taranger a, Elisabet Bergfors a, Valter Sundh a, Teresa Lagerga˚rd b, Erik O8 stergaard c, Helen Cicirello d, Helena Ka¨yhty e b
a The Child Health Center, Vaniljgatan 28, S-424 45 Angered, Sweden The Department of Medical Microbiology and Immunology, Go¨teborg Uni6ersity, S-413 46 Go¨teborg, Sweden c Statens Serum Institut, Artilleri6ej 5, 2300 Copenhagen, Denmark d Baxter Healthcare Corporation, 10150 Old Columbia Road, Columbia, MD 21046, USA e National Public Health Institute, Mannerheimintie 166, FIN-00300 -Helsinki, Finland
Received 6 September 2000; received in revised form 9 May 2001; accepted 22 May 2001
Abstract In an open trial, 400 infants were randomized to vaccination with a combined diphtheria– tetanus– pertussis-inactivated polio vaccine (DTaP-IPV) either mixed with a Haemophilus influenzae type b (Hib) tetanus toxoid conjugate immediately before injection (DTaP-IPV/Hib (mix)) or given concurrently with the Hib conjugate at separate injection sites (DTaP-IPV + Hib (sep)). The pertussis component consisted of pertussis toxoid alone. The vaccines were given intramuscularly at 3, 5 and 12 months of age. No vaccine-related serious adverse events occurred. Local reactions were evaluated from diary cards completed by the parents. Infants who received DTaP-IPV/Hib (mix) experienced fewer local reactions. Sera were obtained 28 – 45 days after the second and third vaccinations. Total Hib capsular antibodies were similar in the two groups after the second injection but lower in the group receiving DTaP-IPV/Hib (mix) than in the group receiving DTaP-IPV + Hib (sep) after the third injection (geometric mean 6.1 vs 10.4 mg/ml). Mixing of the vaccines also led to somewhat lower diphtheria toxin antibodies (5.9 vs 7.7 IU/ml after the third injection) while tetanus antibodies were higher (3.9 vs 2.5 IU/ml after the third injection). Antibodies against pertussis toxin and the three polio virus types were similar in the two groups. The moderate impairment of the Hib antibody response caused by mixing of the Hib conjugate with aluminium adsorbed DTaP may be due to physicochemical interference but is probably of little clinical importance because of the ability of the Hib conjugates to induce an immunologic memory. © 2001 Published by Elsevier Science Ltd. Keywords: Hib conjugate vaccine; Immunogenicity; Reactogenicity; Combination vaccines
1. Introduction With the increasing availability of new vaccines which protect children against severe diseases, the issue of multiple injections have become an important * Corresponding author. Tel.: + 46-31-330-6635; fax: 46-31-3303945. E-mail address: [email protected] (N. Knutsson).
problem in child health care. Much effort has been devoted to developing vaccines which can be administered concurrently in one injection without compromising immunogenicity, safety and tolerability. Combining vaccines against diphtheria, tetanus and pertussis with the recently introduced Haemophilus influenzae type b (Hib) conjugate vaccines has been a particular problem. Several studies have shown that such combinations lead to a more or less pronounced
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decrease in the immunogenicity of the Hib conjugate. This problem has been encountered when the pertussis component of the DTP has been an acellular pertussis vaccine (aP) but not when it has been a whole cell pertussis vaccine (wP) [1– 5]. Acellular pertussis vaccines have widely varying compositions with one to five components. The aim of the present study was to see if a DTaP with the simplest composition of the aP, pertussis toxoid alone, decreased the antibody response to Hib, when the DTaP, which also contained a 3-valent inactivated polio vaccine, and Hib conjugate vaccines were mixed immediately before injection.
2. Subjects and methods
2.1. Study design Healthy term infants were recruited from Child Health Centers in Go¨ teborg. The infants were randomized to one of two groups by opening a sealed envelope when the parents had given consent immediately before vaccination. One group of children received a 4-valent vaccine against diphtheria, tetanus, pertussis and polio in the left leg and the Hib conjugate in the right leg (DTaP-IPV + Hib (sep)). Children in the other group received all vaccines as a single injection in the left leg. In this group, the liquid DTaP-IPV vaccine was the diluent for the lyophilized Hib vaccine just before injection (DTaP-IPV/Hib (mix)). Adverse events, such as temperature, local reactions and systemic complaints, were recorded in diaries by parents at 6, 24 and 48 h after each vaccination. If the temperature was elevated during the first 48 h, the parents continued with daily measurements until the fever resolved. The injection site was inspected every day for 1 week after each vaccination. The information was collected by a study nurse during a structured telephone interview 1 week after each vaccination. The interview also included questions concerning intercurrent illnesses. There were 407 infants enrolled in the study. Of these, 204 were randomized to receive DTaP-IPV/Hib (mix) (116 boys and 88 girls), and 203 to receive DTaP-IPV +Hib (sep) (102 boys and 101 girls). Seven infants were withdrawn before receiving all scheduled doses. In all seven cases the parents changed their minds about participation due to the need to provide blood samples. One of the infants belonged to the DTaP-IPV/Hib (mix) group and six to the DTaPIPV + Hib (sep) group. Structured telephone interviews were made to the parents of all children when 3 and 12 months had elapsed after the third vaccination in order to follow the incidence of pertussis and the occurrence of possible late adverse reactions.
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2.2. Vaccines The diphtheria and tetanus toxoids and the 3-valent inactivated polio vaccine were produced by Statens Serum Institute, Copenhagen, Denmark. The pertussis toxoid was produced by North American Vaccine Inc., Columbia, Maryland, USA. It consisted of pertussis toxin inactivated by hydrogen peroxide. This pertussis toxoid protected children against pertussis in a doubleblind placebo-controlled efficacy trial [6]. Each vaccine dose consisted of 25 flocculation units (Lf) of diphtheria toxoid, 7 Lf of tetanus toxoid and 40 mg of pertussis toxoid. The D-antigen contents of poliovirus types 1, 2 and 3 were 40:8:32 units, respectively. The Hib vaccine was the commercially available Act-HIB® (Pasteur Me´ rieux Connaught, Marcy l’Etoile, France) consisting of 10 mg of Hib capsular polysaccharide conjugated to 24 mg of tetanus toxoid. All vaccines were given intramuscularly into the anterolateral aspect of the thigh using a prefilled single dose syringe with a pre-attached 16 mm needle of 26 gauge. The combined diphtheria, tetanus, pertussis and polio vaccine was adsorbed on to 0.5 mg of aluminium hydroxide. The final volume of all vaccines was 0.5 ml.
2.3. Serological methods Diphtheria toxin neutralizing antibodies were determined by the Vero cell assay [7]. IgG antibodies against tetanus toxoid and pertussis toxin and against the capsular polysaccharide of Hib were determined by enzyme-linked immunosorbent assay (ELISA) at Go¨ teborg University [7,8]. Total Hib antibodies were determined by a radio antigen binding assay (RABA) at the National Public Health Institute, Helsinki [9]. Polio antibodies were determined in duplicate in a neutralization test in cell cultures [10] at Statens Serum Institute, Copenhagen. Serum samples in two-fold dilutions were incubated for 3 h at 37 °C with 100 TCID50 of each poliovirus type. Cells were added and incubation was continued for 7 days at 35 °C. The titer was defined as the fold dilution of serum which neutralized virus in half of the cultures. The minimum levels of detection for the respective methods were 0.01 international units (IU)/ml for diphtheria and tetanus antibodies, 1 unit/ml for pertussis toxin antibodies, 0.2 mg/ml for Hib capsular IgG antibodies, 0.06 mg/ml for total Hib antibodies and a neutralizing titer of 4 for polio antibodies. Sera with undetectable antibodies were arbitrarily assigned half of the level of detection.
2.4. Statistics Serum antibody concentrations between the two groups of subjects were compared using the permutation t-test after logarithmic transformation of data. Proportions were compared with Fisher’s exact test.
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2.5. Ethics
3.1. Ad6erse reactions
The study protocol was approved by the Medical Products Agency, Uppsala, Sweden and the Ethics Committee, Go¨ teborg University. All parents gave written consent after receiving written and oral information about the study.
There were no vaccine-related serious adverse events after any vaccination. Hospitalizations within 30 days post-vaccination occurred among 12 infants. None of them were judged by the clinical investigators to be vaccine-related. Table 1 shows the proportions of children with temperature E 38 °C and E 39 °C within 48 h and 7 days after each vaccination. Significantly more children in the DTaP-IPV/ Hib (mix) group had temperature E38 °C within 48 h of the first vaccination than the children in the DTaP-IPV + Hib (sep) group (13% vs 6%, PB0.05). This difference did not occur after the second and third dose. In both groups the proportion of children with fever E 38 °C increased with the receipt of additional doses.
3. Results A total of 203 children received all three doses of DTaP-IPV/Hib (mix) and 197 children received DTaPIPV +Hib (sep) three times. The mean age at the time of doses 1, 2 and 3 was 96, 159 and 356 days, respectively, in the DTaP-IPV/Hib (mix) group and 95, 157 and 356 days, respectively, in the DTaP-IPV+ Hib (sep) group.
Table 1 Occurrence of fever E38 °C and E39 °C during the first 48 h and 7 days post-vaccination by vaccination number Vaccination
DTaP-IPV/Hib (mix) n/N (%)
DTaP-IPV+Hib (sep) n/N (%)
P-value
Vaccination 1 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
27/204 (13) 2/204 (1)
13/203 (6) 1/203 (0.5)
P= 0.029 P\0.2
Vaccination 2 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
46/204 (23) 8/204 (4)
40/201 (20) 3/201 (1.5)
P\0.2 P\0.2
Vaccination 3 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
49/203 (24) 10/203 (5)
34/197 (17) 6/197 (3)
P= 0.108 P\0.2
Table 2 Local reactions at the injection site within 1 week after each vaccination Hib (right thigh) n/N (%)
DTaP/IPV (left thigh) n/N (%)
DTaP-IPV/Hib (mix) (left thigh) n/N (%)
P-Value left thigh vs left thigh
Vaccination 1 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
8/203 0/203 6/203 0/203
(3.9) (0.0) (3.0) (0.0)
14/203 (6.9) 0/203 (0.0) 17/203 (8.4) 0/203 (0.0)
13/204 (6.4) 0/204 (0.0) 11/204 (5.4) 0/204 (0.0)
P\0.2
Vaccination 2 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
1/201 0/201 1/201 0/201
(0.5) (0.0) (0.5) (0.0)
39/201 (19.4) 0/201 (0.0) 52/201 (25.9) 0/201 (0.0)
22/204 (10.8) 0/204 (0.0) 33/204 (16.2) 1/204 (0.5)
Vaccination 3 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
5/197 0/197 4/197 0/197
(2.5) (0.0) (2.0) (0.0)
49/197 (24.9) 3/197 (1.5) 49/197 (24.9) 1/197 (0.5)
27/203 (13.3) 4/203 (2.0) 33/203 (16.3) 3/203 (1.5)
P\0.2
P =0.018 P= 0.02
P= 0.003 P= 0.03
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Table 3 Antibody concentrations 28–45 days after the second vaccination Antigen
DTaP-IPV/Hib (mix) 1 injection
DTaP-IPV+Hib (sep) 2 injections
Pertussis toxin IgG U/ml Numbers of sera analyzed Geometric mean 95% CI Range
185 126.5 113.1, 141.5 14.0–401.0
188 121.1 109.3, 134.3 10.0–401.0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
184 1.3 1.1, 1.5 0.04–33.2
188 0.7 0.6, 0.9 0.04–23.0
Diphtheria toxin IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
185 0.4 0.4, 0.5 0.01–2.6
187 0.7 0.6, 0.8 0.01–5.1
Hib IgG mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
178 0.4 0.3, 0.5 0.05–22.4
185 0.6 0.4, 0.7 0.05–35.1
Hib total antibodies mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
174 0.6 0.5, 0.7 0.06–26.1
183 0.7 0.6, 0.9 0.06–44.3
Polio-1 titers Numbers of sera analyzed Geometric mean 95% CI Range
178 621.4 475.3, 812.4 B4–5790.0
178 774.3 607.7, 986.6 11.0–5790.0
Polio-2 titers Numbers of sera analyzed Geometric mean 95% CI Range
178 1097.2 858.0, 1403.1 16.0–5790.0
178 1190.5 954.0, 1485.8 8.0–5790.0
Polio-3 titres Numbers of sera analyzed Geometric mean 95% CI Range
177 499.2 378.3, 658.8 B4–5790.0
178 668.8 516.8, 865.6 11.0–5790.0
Table 2 shows local reactions within 1 week after each vaccination. Local reactions increased with the numbers of doses received in both groups. DTaPIPV induced significantly more redness and swelling than DTaP-IPV/Hib (mix) after the second and third injections (comparisons of left vs left thighs in Table 2). Parents of 396 and 400 participating children, respectively, were interviewed 3 and 12 months after the third vaccination. All children were healthy and none had experienced any events which could be interpreted as a reaction to the vaccine.
P-value
P\0.2
PB0.0001
P= 0.0006
P =0.08
P =0.12
P\0.2
P\0.2
P=0.16
3.2. Antibody concentrations A total of 403 blood samples were obtained after the second and 396 after the third vaccination. Of these, 30 samples obtained after the second vaccination and 15 obtained after the third vaccination were not included in the analysis because they were obtained within less than 28 or more than 45 days after the second and third vaccination, respectively. Tables 3 and 4 show antibody concentrations for all sera obtained within 28–45 days after the second and third vaccinations. Table 5 shows the number of infants with antibody levels below levels
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usually considered related to protection. For pertussis antibodies an arbitrary level of 10 units/ml was used. Figs. 1–3 show reverse cumulative distribution curves for diphtheria, tetanus and total Hib antibodies after the third injection. There were no significant differences between the two study groups with regards to pertussis toxin antibodies or for antibodies against any of the three types of polio virus. Hib IgG (ELISA) and total Hib (RABA) were similar in the two groups after the second injection. After the third injection both Hib IgG and total Hib antibodies were significantly lower in the DTaP-IPV/Hib (mix) group, but in both groups all
children had total antibody concentrations above 0.15 mg/ml (correlating with short-term protection) after the third vaccination. Diphtheria antibodies were significanly lower and tetanus antibodies significantly higher in the DTaP-IPV/Hib (mix) group than in the DTaPIPV + Hib (sep) group after both the second and the third vaccination. All children attained long-term protective levels (\ 0.1 IU/ml) for tetanus after three doses. For diphtheria, 100% and 94% in the DTaP-IPV/ Hib (mix) and DTaP-IPV+ Hib (sep) groups, respectively, achieved long term protective levels (\0.1 IU/ml) after three doses.
Table 4 Antibody concentrations 28–45 days after the third vaccination Antigen
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
Pertussis toxin IgG U/ml Numbers of sera analyzed Geometric mean 95% CI Range
192 260.9 243.3, 279.8 36.0–401.0
185 235.8 217.3, 256.0 40.0–401.0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
191 3.9 3.4, 4.4 0.2–36.4
187 2.5 2.2, 2.9 0.2–30.8
Diphtheria toxin IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
190 5.9 5.0, 6.9 0.2–29.0
184 7.7 6.5, 9.3 0.01–29.0
Hib IgG mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
190 6.9 5.7, 8.3 0.05–211.6
186 11.3 8.9, 14.3 0.05–363.2
Hib total antibodies mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
189 6.1 5.2, 7.1 0.3–143.4
185 10.4 8.6, 12.5 0.23–261.1
Polio-1 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 3764.5 3384.5, 4187.0 32.0–5790.0
182 3994.1 3681.4, 4333.4 512.0–5790.0
Polio-2 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 4132.1 3775.5, 4522.5 128.0–5790.0
182 4149.2 3820.8, 4505.8 256.0–5790.0
Polio-3 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 2964.6 2543.0, 3456.2 23.0–5790.0
182 3332.4 2910.6, 3815.2 32.0–5790.0
P-value
P\0.2
PB0.0001
P= 0.02
PB0.001
PB0.0001
P\0.2
P\0.2
P= 0.2
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Table 5 Numbers of children (%) with serum antibody levels below levels related to short- and long-term protection after the second (serum sample 1) and third (serum sample 2) vaccination Serum sample 1 Antigen
Serum sample 2
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
Pertussis toxin IgG U/ml Numbers of sera analyzed B10
185 0
188 0
192 0
185 0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed B0.01 B0.1
191 0 3 (1.6)
187 0 7 (3.7)
191 0 0
187 0 0
Diphtheria toxin IU/ml Number of sera analyzed B0.01 B0.1
190 0 22 (12)
184 0 16 (9)
190 0 0
184 0 3 (1.6)
Hib IgG mg/ml Numbers of sera analyzed B0.2 B1.0
190 42 (22) 139 (73)
186 31 (17) 137 (74)
191 1 (0.5) 15 (8)
186 3 (1.6) 15 (8)
Hib total antibodies mg/ml Numbers of sera analyzed B0.15 B1.0
189 8 (4.2) 132 (70)
185 13 (7) 131 (71)
189 0 8 (4.2)
185 0 7 (3.8)
Polio-1 titers Numbers of sera analyzed B4 B16
186 1 (0.5) 4 (2.2)
182 0 1 (0.5)
186 0 0
182 0 0
Polio-2 titers Numbers of sera analyzed B4 B16
186 0 0
182 0 2 (1.1)
186 0 0
182 0 0
Polio-3 titers Numbers of sera analyzed B4 B16
186 1 (0.5) 5 (2.7)
182 0 1 (0.5)
186 0 0
182 0 0
4. Discussion This study demonstrated that the combined vaccine with diphtheria, tetanus and pertussis toxoids and inactivated polio virus vaccine was safe when given concurrently with a Hib conjugate vaccine, whether given as separate injections or mixed in the syringe immediately before injection. No vaccine-related serious adverse events occurred in any of the 400 participating children who received all three scheduled vaccinations and all children were healthy according to telephone interviews with their parents when more than 3 and 12 months, respectively, had elapsed after the third vaccination. Mixing of the DTaP-IPV with the Hib conjugate in the syringe immediately before injection surprisingly gave rise to a lower incidence of pronounced local reactions after all injections than administration of
the DTaP-IPV alone. This was not the case in other studies in which DTaP or DTaP-IPV vaccines were mixed with Hib conjugates [2,4,5].
Fig. 1. Reverse cumulative distribution curve for diphtheria toxin antibodies after the third injection.
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Fig. 2. Reverse cumulative distribution curve for tetanus toxoid antibodies after the third injection.
Fig. 3. Reverse cumulative distribution curve for total Haemophilus influenzae type b antibodies as measured by RABA after the third injection.
The study showed that the DTaP vaccine used in the present study, in which the pertussis component consisted only of pertussis toxoid, in similarity with DTaP with two- and three component acellular pertussis vaccines, caused an impairment of the antibody response to the Hib conjugate vaccine [2,4,5]. This suppression of the Hib antibody response, which was seen both for total antibodies as measured by RABA and for IgG antibodies as measured by ELISA, was moderate and seen only after the third injection. RABA is based on precipitation of antipolysaccharide antibodies and measures total amount of precipitating antibodies (IgG, IgM and IgA). ELISA is based on binding of antibodies to solid face attached polysaccharide and enables assays of different immunoglobulin classes. The affinity of antibodies may contribute to the differences in results obtained by these two methods and ELISA possibly overestimates low-affinity antibodies. After the
third injection, the geometric mean Hib antibodies in the DTaP-IPV/Hib (mix) group was 59% (total antibodies) and 61% (IgG) of the concentrations in the DTaP-IPV +Hib (sep) group. This is similar to or less pronounced than seen after mixing of other DTaP vaccines with Hib conjugates [2,4,5]. The impairment of the Hib antibody response induced by mixing with DTaP vaccines is considered to be due to physicochemical interference in the syringe [3]. The possibility that the aluminium content of the DTaP affects the Hib antibody response should be considered, since it has been shown that an aluminium adsorbed Hib tetanus toxoid conjugate had a poor immunogenicity as compared to the same nonadsorbed vaccine [11]. According to a recent review, the moderate impairment of the Hib antibody response related to mixing of the Hib conjugate with DTaP should be considered of little clinical importance [3]. Previous estimates of protective levels of Hib serum antibodies are deemed to be of less relevance for the Hib serum antibodies induced by conjugated vaccines. Instead the ability of the Hib conjugates to induce an immunologic memory is considered of major importance for subsequent protection against invasive Hib disease [3]. Mixing of the DTaP-IPV vaccine with the Hib conjugate led to a small but significant impairment of the diphtheria toxin antibody response. This may be due to physicochemical interference in the syringe. Other studies of mixing of vaccines immediately before injection have also shown tendencies to lower antibody concentrations to many vaccine antigens when vaccines are mixed [1,2,4,5]. Surprisingly, the present study showed a significantly higher response to the tetanus component when the DTaP-IPV was mixed with the Hibtetanus toxoid conjugate before injection than when the vaccines were given at separate injection sites. This difference was significant both after the second and third vaccination. Another study, using the same Hib tetanus toxoid conjugate, did not show a similar difference [1]. We have no explanation for this finding. In conclusion, mixing of DTaP-IPV (in which the pertussis vaccine consisted only of pertussis toxoid) with a Hib conjugate vaccine led to an impaired antibody response to the Hib polysaccharide, but, since protective levels of anti-Hib polysaccharide antibodies were achieved by all subjects, the clinical relevance of this is probably of little clinical importance.
Acknowledgements We thank all the nurses for taking good care of the families, performing the vaccinations and obtaining blood samples, Maja Berg and Eva Gunnarsson for skilful technical assistance and Rose-Marie Carlsson for help with construction of the reverse cumulative distri-
N. Knutsson et al. / Vaccine 19 (2001) 4396–4403
bution curves. The study was supported by North American Vaccine Inc., Columbia, Maryland, USA, now acquired by Baxter Healthcare Corporation.
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Immunogenicity and reactogenicity of diphtheria, tetanus and pertussis toxoids combined with inactivated polio vaccine, when administered concomitantly with or as a diluent for a Hib conjugate vaccine Nina Knutsson a,*, Birger Trollfors a, John Taranger a, Elisabet Bergfors a, Valter Sundh a, Teresa Lagerga˚rd b, Erik O8 stergaard c, Helen Cicirello d, Helena Ka¨yhty e b
a The Child Health Center, Vaniljgatan 28, S-424 45 Angered, Sweden The Department of Medical Microbiology and Immunology, Go¨teborg Uni6ersity, S-413 46 Go¨teborg, Sweden c Statens Serum Institut, Artilleri6ej 5, 2300 Copenhagen, Denmark d Baxter Healthcare Corporation, 10150 Old Columbia Road, Columbia, MD 21046, USA e National Public Health Institute, Mannerheimintie 166, FIN-00300 -Helsinki, Finland
Received 6 September 2000; received in revised form 9 May 2001; accepted 22 May 2001
Abstract In an open trial, 400 infants were randomized to vaccination with a combined diphtheria– tetanus– pertussis-inactivated polio vaccine (DTaP-IPV) either mixed with a Haemophilus influenzae type b (Hib) tetanus toxoid conjugate immediately before injection (DTaP-IPV/Hib (mix)) or given concurrently with the Hib conjugate at separate injection sites (DTaP-IPV + Hib (sep)). The pertussis component consisted of pertussis toxoid alone. The vaccines were given intramuscularly at 3, 5 and 12 months of age. No vaccine-related serious adverse events occurred. Local reactions were evaluated from diary cards completed by the parents. Infants who received DTaP-IPV/Hib (mix) experienced fewer local reactions. Sera were obtained 28 – 45 days after the second and third vaccinations. Total Hib capsular antibodies were similar in the two groups after the second injection but lower in the group receiving DTaP-IPV/Hib (mix) than in the group receiving DTaP-IPV + Hib (sep) after the third injection (geometric mean 6.1 vs 10.4 mg/ml). Mixing of the vaccines also led to somewhat lower diphtheria toxin antibodies (5.9 vs 7.7 IU/ml after the third injection) while tetanus antibodies were higher (3.9 vs 2.5 IU/ml after the third injection). Antibodies against pertussis toxin and the three polio virus types were similar in the two groups. The moderate impairment of the Hib antibody response caused by mixing of the Hib conjugate with aluminium adsorbed DTaP may be due to physicochemical interference but is probably of little clinical importance because of the ability of the Hib conjugates to induce an immunologic memory. © 2001 Published by Elsevier Science Ltd. Keywords: Hib conjugate vaccine; Immunogenicity; Reactogenicity; Combination vaccines
1. Introduction With the increasing availability of new vaccines which protect children against severe diseases, the issue of multiple injections have become an important * Corresponding author. Tel.: + 46-31-330-6635; fax: 46-31-3303945. E-mail address: [email protected] (N. Knutsson).
problem in child health care. Much effort has been devoted to developing vaccines which can be administered concurrently in one injection without compromising immunogenicity, safety and tolerability. Combining vaccines against diphtheria, tetanus and pertussis with the recently introduced Haemophilus influenzae type b (Hib) conjugate vaccines has been a particular problem. Several studies have shown that such combinations lead to a more or less pronounced
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decrease in the immunogenicity of the Hib conjugate. This problem has been encountered when the pertussis component of the DTP has been an acellular pertussis vaccine (aP) but not when it has been a whole cell pertussis vaccine (wP) [1– 5]. Acellular pertussis vaccines have widely varying compositions with one to five components. The aim of the present study was to see if a DTaP with the simplest composition of the aP, pertussis toxoid alone, decreased the antibody response to Hib, when the DTaP, which also contained a 3-valent inactivated polio vaccine, and Hib conjugate vaccines were mixed immediately before injection.
2. Subjects and methods
2.1. Study design Healthy term infants were recruited from Child Health Centers in Go¨ teborg. The infants were randomized to one of two groups by opening a sealed envelope when the parents had given consent immediately before vaccination. One group of children received a 4-valent vaccine against diphtheria, tetanus, pertussis and polio in the left leg and the Hib conjugate in the right leg (DTaP-IPV + Hib (sep)). Children in the other group received all vaccines as a single injection in the left leg. In this group, the liquid DTaP-IPV vaccine was the diluent for the lyophilized Hib vaccine just before injection (DTaP-IPV/Hib (mix)). Adverse events, such as temperature, local reactions and systemic complaints, were recorded in diaries by parents at 6, 24 and 48 h after each vaccination. If the temperature was elevated during the first 48 h, the parents continued with daily measurements until the fever resolved. The injection site was inspected every day for 1 week after each vaccination. The information was collected by a study nurse during a structured telephone interview 1 week after each vaccination. The interview also included questions concerning intercurrent illnesses. There were 407 infants enrolled in the study. Of these, 204 were randomized to receive DTaP-IPV/Hib (mix) (116 boys and 88 girls), and 203 to receive DTaP-IPV +Hib (sep) (102 boys and 101 girls). Seven infants were withdrawn before receiving all scheduled doses. In all seven cases the parents changed their minds about participation due to the need to provide blood samples. One of the infants belonged to the DTaP-IPV/Hib (mix) group and six to the DTaPIPV + Hib (sep) group. Structured telephone interviews were made to the parents of all children when 3 and 12 months had elapsed after the third vaccination in order to follow the incidence of pertussis and the occurrence of possible late adverse reactions.
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2.2. Vaccines The diphtheria and tetanus toxoids and the 3-valent inactivated polio vaccine were produced by Statens Serum Institute, Copenhagen, Denmark. The pertussis toxoid was produced by North American Vaccine Inc., Columbia, Maryland, USA. It consisted of pertussis toxin inactivated by hydrogen peroxide. This pertussis toxoid protected children against pertussis in a doubleblind placebo-controlled efficacy trial [6]. Each vaccine dose consisted of 25 flocculation units (Lf) of diphtheria toxoid, 7 Lf of tetanus toxoid and 40 mg of pertussis toxoid. The D-antigen contents of poliovirus types 1, 2 and 3 were 40:8:32 units, respectively. The Hib vaccine was the commercially available Act-HIB® (Pasteur Me´ rieux Connaught, Marcy l’Etoile, France) consisting of 10 mg of Hib capsular polysaccharide conjugated to 24 mg of tetanus toxoid. All vaccines were given intramuscularly into the anterolateral aspect of the thigh using a prefilled single dose syringe with a pre-attached 16 mm needle of 26 gauge. The combined diphtheria, tetanus, pertussis and polio vaccine was adsorbed on to 0.5 mg of aluminium hydroxide. The final volume of all vaccines was 0.5 ml.
2.3. Serological methods Diphtheria toxin neutralizing antibodies were determined by the Vero cell assay [7]. IgG antibodies against tetanus toxoid and pertussis toxin and against the capsular polysaccharide of Hib were determined by enzyme-linked immunosorbent assay (ELISA) at Go¨ teborg University [7,8]. Total Hib antibodies were determined by a radio antigen binding assay (RABA) at the National Public Health Institute, Helsinki [9]. Polio antibodies were determined in duplicate in a neutralization test in cell cultures [10] at Statens Serum Institute, Copenhagen. Serum samples in two-fold dilutions were incubated for 3 h at 37 °C with 100 TCID50 of each poliovirus type. Cells were added and incubation was continued for 7 days at 35 °C. The titer was defined as the fold dilution of serum which neutralized virus in half of the cultures. The minimum levels of detection for the respective methods were 0.01 international units (IU)/ml for diphtheria and tetanus antibodies, 1 unit/ml for pertussis toxin antibodies, 0.2 mg/ml for Hib capsular IgG antibodies, 0.06 mg/ml for total Hib antibodies and a neutralizing titer of 4 for polio antibodies. Sera with undetectable antibodies were arbitrarily assigned half of the level of detection.
2.4. Statistics Serum antibody concentrations between the two groups of subjects were compared using the permutation t-test after logarithmic transformation of data. Proportions were compared with Fisher’s exact test.
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2.5. Ethics
3.1. Ad6erse reactions
The study protocol was approved by the Medical Products Agency, Uppsala, Sweden and the Ethics Committee, Go¨ teborg University. All parents gave written consent after receiving written and oral information about the study.
There were no vaccine-related serious adverse events after any vaccination. Hospitalizations within 30 days post-vaccination occurred among 12 infants. None of them were judged by the clinical investigators to be vaccine-related. Table 1 shows the proportions of children with temperature E 38 °C and E 39 °C within 48 h and 7 days after each vaccination. Significantly more children in the DTaP-IPV/ Hib (mix) group had temperature E38 °C within 48 h of the first vaccination than the children in the DTaP-IPV + Hib (sep) group (13% vs 6%, PB0.05). This difference did not occur after the second and third dose. In both groups the proportion of children with fever E 38 °C increased with the receipt of additional doses.
3. Results A total of 203 children received all three doses of DTaP-IPV/Hib (mix) and 197 children received DTaPIPV +Hib (sep) three times. The mean age at the time of doses 1, 2 and 3 was 96, 159 and 356 days, respectively, in the DTaP-IPV/Hib (mix) group and 95, 157 and 356 days, respectively, in the DTaP-IPV+ Hib (sep) group.
Table 1 Occurrence of fever E38 °C and E39 °C during the first 48 h and 7 days post-vaccination by vaccination number Vaccination
DTaP-IPV/Hib (mix) n/N (%)
DTaP-IPV+Hib (sep) n/N (%)
P-value
Vaccination 1 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
27/204 (13) 2/204 (1)
13/203 (6) 1/203 (0.5)
P= 0.029 P\0.2
Vaccination 2 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
46/204 (23) 8/204 (4)
40/201 (20) 3/201 (1.5)
P\0.2 P\0.2
Vaccination 3 TemperatureE38 °C within 48 h TemperatureE39 °C within 7 days
49/203 (24) 10/203 (5)
34/197 (17) 6/197 (3)
P= 0.108 P\0.2
Table 2 Local reactions at the injection site within 1 week after each vaccination Hib (right thigh) n/N (%)
DTaP/IPV (left thigh) n/N (%)
DTaP-IPV/Hib (mix) (left thigh) n/N (%)
P-Value left thigh vs left thigh
Vaccination 1 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
8/203 0/203 6/203 0/203
(3.9) (0.0) (3.0) (0.0)
14/203 (6.9) 0/203 (0.0) 17/203 (8.4) 0/203 (0.0)
13/204 (6.4) 0/204 (0.0) 11/204 (5.4) 0/204 (0.0)
P\0.2
Vaccination 2 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
1/201 0/201 1/201 0/201
(0.5) (0.0) (0.5) (0.0)
39/201 (19.4) 0/201 (0.0) 52/201 (25.9) 0/201 (0.0)
22/204 (10.8) 0/204 (0.0) 33/204 (16.2) 1/204 (0.5)
Vaccination 3 RednessE2 cm RednessE6 cm SwellingE2 cm SwellingE6 cm
5/197 0/197 4/197 0/197
(2.5) (0.0) (2.0) (0.0)
49/197 (24.9) 3/197 (1.5) 49/197 (24.9) 1/197 (0.5)
27/203 (13.3) 4/203 (2.0) 33/203 (16.3) 3/203 (1.5)
P\0.2
P =0.018 P= 0.02
P= 0.003 P= 0.03
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Table 3 Antibody concentrations 28–45 days after the second vaccination Antigen
DTaP-IPV/Hib (mix) 1 injection
DTaP-IPV+Hib (sep) 2 injections
Pertussis toxin IgG U/ml Numbers of sera analyzed Geometric mean 95% CI Range
185 126.5 113.1, 141.5 14.0–401.0
188 121.1 109.3, 134.3 10.0–401.0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
184 1.3 1.1, 1.5 0.04–33.2
188 0.7 0.6, 0.9 0.04–23.0
Diphtheria toxin IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
185 0.4 0.4, 0.5 0.01–2.6
187 0.7 0.6, 0.8 0.01–5.1
Hib IgG mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
178 0.4 0.3, 0.5 0.05–22.4
185 0.6 0.4, 0.7 0.05–35.1
Hib total antibodies mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
174 0.6 0.5, 0.7 0.06–26.1
183 0.7 0.6, 0.9 0.06–44.3
Polio-1 titers Numbers of sera analyzed Geometric mean 95% CI Range
178 621.4 475.3, 812.4 B4–5790.0
178 774.3 607.7, 986.6 11.0–5790.0
Polio-2 titers Numbers of sera analyzed Geometric mean 95% CI Range
178 1097.2 858.0, 1403.1 16.0–5790.0
178 1190.5 954.0, 1485.8 8.0–5790.0
Polio-3 titres Numbers of sera analyzed Geometric mean 95% CI Range
177 499.2 378.3, 658.8 B4–5790.0
178 668.8 516.8, 865.6 11.0–5790.0
Table 2 shows local reactions within 1 week after each vaccination. Local reactions increased with the numbers of doses received in both groups. DTaPIPV induced significantly more redness and swelling than DTaP-IPV/Hib (mix) after the second and third injections (comparisons of left vs left thighs in Table 2). Parents of 396 and 400 participating children, respectively, were interviewed 3 and 12 months after the third vaccination. All children were healthy and none had experienced any events which could be interpreted as a reaction to the vaccine.
P-value
P\0.2
PB0.0001
P= 0.0006
P =0.08
P =0.12
P\0.2
P\0.2
P=0.16
3.2. Antibody concentrations A total of 403 blood samples were obtained after the second and 396 after the third vaccination. Of these, 30 samples obtained after the second vaccination and 15 obtained after the third vaccination were not included in the analysis because they were obtained within less than 28 or more than 45 days after the second and third vaccination, respectively. Tables 3 and 4 show antibody concentrations for all sera obtained within 28–45 days after the second and third vaccinations. Table 5 shows the number of infants with antibody levels below levels
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usually considered related to protection. For pertussis antibodies an arbitrary level of 10 units/ml was used. Figs. 1–3 show reverse cumulative distribution curves for diphtheria, tetanus and total Hib antibodies after the third injection. There were no significant differences between the two study groups with regards to pertussis toxin antibodies or for antibodies against any of the three types of polio virus. Hib IgG (ELISA) and total Hib (RABA) were similar in the two groups after the second injection. After the third injection both Hib IgG and total Hib antibodies were significantly lower in the DTaP-IPV/Hib (mix) group, but in both groups all
children had total antibody concentrations above 0.15 mg/ml (correlating with short-term protection) after the third vaccination. Diphtheria antibodies were significanly lower and tetanus antibodies significantly higher in the DTaP-IPV/Hib (mix) group than in the DTaPIPV + Hib (sep) group after both the second and the third vaccination. All children attained long-term protective levels (\ 0.1 IU/ml) for tetanus after three doses. For diphtheria, 100% and 94% in the DTaP-IPV/ Hib (mix) and DTaP-IPV+ Hib (sep) groups, respectively, achieved long term protective levels (\0.1 IU/ml) after three doses.
Table 4 Antibody concentrations 28–45 days after the third vaccination Antigen
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
Pertussis toxin IgG U/ml Numbers of sera analyzed Geometric mean 95% CI Range
192 260.9 243.3, 279.8 36.0–401.0
185 235.8 217.3, 256.0 40.0–401.0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
191 3.9 3.4, 4.4 0.2–36.4
187 2.5 2.2, 2.9 0.2–30.8
Diphtheria toxin IU/ml Numbers of sera analyzed Geometric mean 95% CI Range
190 5.9 5.0, 6.9 0.2–29.0
184 7.7 6.5, 9.3 0.01–29.0
Hib IgG mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
190 6.9 5.7, 8.3 0.05–211.6
186 11.3 8.9, 14.3 0.05–363.2
Hib total antibodies mg/ml Numbers of sera analyzed Geometric mean 95% CI Range
189 6.1 5.2, 7.1 0.3–143.4
185 10.4 8.6, 12.5 0.23–261.1
Polio-1 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 3764.5 3384.5, 4187.0 32.0–5790.0
182 3994.1 3681.4, 4333.4 512.0–5790.0
Polio-2 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 4132.1 3775.5, 4522.5 128.0–5790.0
182 4149.2 3820.8, 4505.8 256.0–5790.0
Polio-3 titers Numbers of sera analyzed Geometric mean 95% CI Range
186 2964.6 2543.0, 3456.2 23.0–5790.0
182 3332.4 2910.6, 3815.2 32.0–5790.0
P-value
P\0.2
PB0.0001
P= 0.02
PB0.001
PB0.0001
P\0.2
P\0.2
P= 0.2
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Table 5 Numbers of children (%) with serum antibody levels below levels related to short- and long-term protection after the second (serum sample 1) and third (serum sample 2) vaccination Serum sample 1 Antigen
Serum sample 2
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
DTaP-IPV/Hib (mix)
DTaP-IPV+Hib (sep)
Pertussis toxin IgG U/ml Numbers of sera analyzed B10
185 0
188 0
192 0
185 0
Tetanus toxoid IgG IU/ml Numbers of sera analyzed B0.01 B0.1
191 0 3 (1.6)
187 0 7 (3.7)
191 0 0
187 0 0
Diphtheria toxin IU/ml Number of sera analyzed B0.01 B0.1
190 0 22 (12)
184 0 16 (9)
190 0 0
184 0 3 (1.6)
Hib IgG mg/ml Numbers of sera analyzed B0.2 B1.0
190 42 (22) 139 (73)
186 31 (17) 137 (74)
191 1 (0.5) 15 (8)
186 3 (1.6) 15 (8)
Hib total antibodies mg/ml Numbers of sera analyzed B0.15 B1.0
189 8 (4.2) 132 (70)
185 13 (7) 131 (71)
189 0 8 (4.2)
185 0 7 (3.8)
Polio-1 titers Numbers of sera analyzed B4 B16
186 1 (0.5) 4 (2.2)
182 0 1 (0.5)
186 0 0
182 0 0
Polio-2 titers Numbers of sera analyzed B4 B16
186 0 0
182 0 2 (1.1)
186 0 0
182 0 0
Polio-3 titers Numbers of sera analyzed B4 B16
186 1 (0.5) 5 (2.7)
182 0 1 (0.5)
186 0 0
182 0 0
4. Discussion This study demonstrated that the combined vaccine with diphtheria, tetanus and pertussis toxoids and inactivated polio virus vaccine was safe when given concurrently with a Hib conjugate vaccine, whether given as separate injections or mixed in the syringe immediately before injection. No vaccine-related serious adverse events occurred in any of the 400 participating children who received all three scheduled vaccinations and all children were healthy according to telephone interviews with their parents when more than 3 and 12 months, respectively, had elapsed after the third vaccination. Mixing of the DTaP-IPV with the Hib conjugate in the syringe immediately before injection surprisingly gave rise to a lower incidence of pronounced local reactions after all injections than administration of
the DTaP-IPV alone. This was not the case in other studies in which DTaP or DTaP-IPV vaccines were mixed with Hib conjugates [2,4,5].
Fig. 1. Reverse cumulative distribution curve for diphtheria toxin antibodies after the third injection.
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Fig. 2. Reverse cumulative distribution curve for tetanus toxoid antibodies after the third injection.
Fig. 3. Reverse cumulative distribution curve for total Haemophilus influenzae type b antibodies as measured by RABA after the third injection.
The study showed that the DTaP vaccine used in the present study, in which the pertussis component consisted only of pertussis toxoid, in similarity with DTaP with two- and three component acellular pertussis vaccines, caused an impairment of the antibody response to the Hib conjugate vaccine [2,4,5]. This suppression of the Hib antibody response, which was seen both for total antibodies as measured by RABA and for IgG antibodies as measured by ELISA, was moderate and seen only after the third injection. RABA is based on precipitation of antipolysaccharide antibodies and measures total amount of precipitating antibodies (IgG, IgM and IgA). ELISA is based on binding of antibodies to solid face attached polysaccharide and enables assays of different immunoglobulin classes. The affinity of antibodies may contribute to the differences in results obtained by these two methods and ELISA possibly overestimates low-affinity antibodies. After the
third injection, the geometric mean Hib antibodies in the DTaP-IPV/Hib (mix) group was 59% (total antibodies) and 61% (IgG) of the concentrations in the DTaP-IPV +Hib (sep) group. This is similar to or less pronounced than seen after mixing of other DTaP vaccines with Hib conjugates [2,4,5]. The impairment of the Hib antibody response induced by mixing with DTaP vaccines is considered to be due to physicochemical interference in the syringe [3]. The possibility that the aluminium content of the DTaP affects the Hib antibody response should be considered, since it has been shown that an aluminium adsorbed Hib tetanus toxoid conjugate had a poor immunogenicity as compared to the same nonadsorbed vaccine [11]. According to a recent review, the moderate impairment of the Hib antibody response related to mixing of the Hib conjugate with DTaP should be considered of little clinical importance [3]. Previous estimates of protective levels of Hib serum antibodies are deemed to be of less relevance for the Hib serum antibodies induced by conjugated vaccines. Instead the ability of the Hib conjugates to induce an immunologic memory is considered of major importance for subsequent protection against invasive Hib disease [3]. Mixing of the DTaP-IPV vaccine with the Hib conjugate led to a small but significant impairment of the diphtheria toxin antibody response. This may be due to physicochemical interference in the syringe. Other studies of mixing of vaccines immediately before injection have also shown tendencies to lower antibody concentrations to many vaccine antigens when vaccines are mixed [1,2,4,5]. Surprisingly, the present study showed a significantly higher response to the tetanus component when the DTaP-IPV was mixed with the Hibtetanus toxoid conjugate before injection than when the vaccines were given at separate injection sites. This difference was significant both after the second and third vaccination. Another study, using the same Hib tetanus toxoid conjugate, did not show a similar difference [1]. We have no explanation for this finding. In conclusion, mixing of DTaP-IPV (in which the pertussis vaccine consisted only of pertussis toxoid) with a Hib conjugate vaccine led to an impaired antibody response to the Hib polysaccharide, but, since protective levels of anti-Hib polysaccharide antibodies were achieved by all subjects, the clinical relevance of this is probably of little clinical importance.
Acknowledgements We thank all the nurses for taking good care of the families, performing the vaccinations and obtaining blood samples, Maja Berg and Eva Gunnarsson for skilful technical assistance and Rose-Marie Carlsson for help with construction of the reverse cumulative distri-
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bution curves. The study was supported by North American Vaccine Inc., Columbia, Maryland, USA, now acquired by Baxter Healthcare Corporation.
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