Combination vaccines improve compliance with childhood immunization schedules and reduce the number of injections.1–6 There are also programmatic benefits including reduced administration costs, easier record keeping and improved on-time completion of doses.7–9 The immunogenicity of combination vaccines must be noninferior to the vaccine antigens given separately and there should not be increased adverse events associated with providing the vaccines as a combination.
In many countries, infants are vaccinated against diphtheria, tetanus, polio, pertussis, hepatitis B and invasive bacterial infections caused by Haemophilus influenzae type b (Hib), Streptococcus pneumoniae and Neisseria meningitidis type C. A vaccine against type B meningococcus will soon be available in the European Union10 and elsewhere. With the development of new vaccines targeting infants, combination vaccines also “make room” for the implementation of additional vaccines and control of new diseases that affect young children. A hexavalent vaccine that protects against diphtheria, tetanus, pertussis, polio, Hib and hepatitis B has been available in the European Union and Canada for several years. An investigational hexavalent vaccine, DTaP5-IPV-Hib-HepB, comprising diphtheria and tetanus toxoids, 5-component acellular pertussis vaccine, trivalent inactivated poliovirus vaccine (IPV), hepatitis B vaccine (HepB) and Hib polyribosylribitol phosphate capsular polysaccharide conjugated to N. meningitidis outer membrane proteins (PRP-OMP) given concurrently with the 7-valent, pneumococcal conjugate vaccine (PCV7) was well-tolerated and demonstrated strong antibody responses against all vaccine antigens in the primary series given at 2, 4 and 6 months of age.11 The antibody responses and adverse event profiles after the primary series of hexavalent vaccine with concomitant PCV7 were similar when PCV7 was given 1 month after the DTaP5-IPV-Hib-HepB doses and also similar to the marketed, pentavalent DTaP5-IPV/Hib with the HepB given as a separate injection. We now report the safety and immunogenicity of DTaP5-IPV-Hib-HepB coadministered with PCV7 as a booster dose following the primary series in these toddlers at 15 months of age. The overall objective of the study was to evaluate the safety and immunogenicity of the DTaP5-IPV-Hib-HepB candidate vaccine. The control vaccines were selected as representative examples of the standard of care in Canada at the time the study was conducted.
The investigational vaccine was a liquid, hexavalent, preservative-free, diphtheria and tetanus toxoids, 5-component acellular pertussis, trivalent inactivated poliovirus, Hib conjugate, hepatitis B combination vaccine (DTaP5-IPV-Hib-HepB; Sanofi Pasteur, Swiftwater, PA; Merck & Co., Whitehouse Station, NJ). Commercially available, pentavalent DTaP5-IPV/Hib vaccine (Pentacel; Sanofi Pasteur, Toronto, ON, Canada), HepB vaccine (Engerix-B; GlaxoSmithKline, Research Triangle Park, NC) and PCV7 ( Prevnar, Pfizer Inc., New York, NY) were also used (Table 1).
Participants were toddlers who received their 15-month DTaP5-IPV-Hib-HepB booster dose in this study. These participants had received their 3-dose primary series with the same vaccine; the primary series results have already been reported.11 At the time of the initial enrollment, participants were healthy 2-month-olds (42–89 days) born at >37 weeks gestation. Exclusions at the time of enrollment were known or suspected systemic hypersensitivity to any vaccine component; a personal or immediate family history of congenital or acquired immunodeficiency; received immunosuppressive therapy; received blood or blood-derived products; chronic illness that could interfere with trial conduct or completion; any vaccination preceding the first trial vaccination or planned in the 4 weeks after any trial vaccination; coagulation disorder contraindicating intramuscular vaccination; developmental delay or neurological disorder; documented seropositivity to hepatitis B surface antigen (HBsAg) in participant or the participant’s mother or history of Hib, hepatitis B, diphtheria, tetanus, pertussis or poliovirus disease.
Study Design and Procedures
This was a randomized, open-label, phase 2b clinical trial conducted at 8 sites in Canada. The study was approved by the Research Ethics Board at each study site; written informed consent for the entire duration of the study (primary series and toddler dose) was provided by the parent/legal guardian of all participants before any study procedure. The trial was conducted in compliance with the Declaration of Helsinki.
At study entry, infants were randomly allocated via a central, computer-generated algorithm (1:1:1 ratio with a block size of 9) to receive DTaP5-IPV-Hib-HepB administered concomitantly with PCV7 (Group A), DTaP5-IPV-Hib-HepB followed by PCV7 1 month later (Group B) and DTaP5-IPV/Hib plus HepB plus PCV7 administered concomitantly (Group C). Participants retained their infant randomization allocation for the toddler dose; participants in Group A and Group C received all vaccinations at 15 months of age and participants in Group B received DTaP5-IPV-Hib-HepB at 15 months and PCV7 at 16 months of age. DTaP5-IPV-Hib-HepB (0.5 mL intramuscular [IM)] was given in the left thigh, DTaP5-IPV/Hib (0.5 mL IM) was given in the right thigh, PCV7 (0.5 mL IM) was given in the right (Group A and B) or left (Group C) thigh and HepB (0.5 mL IM) was given in the right thigh. Participants were observed for 30 minutes after each injection for immediate reactions. Parents recorded solicited injection-site reactions, daily temperature and solicited and unsolicited systemic adverse events between Day 0 and Day 7 after each concomitantly administered vaccination; no adverse events were collected after PCV7 given at a separate visit.
Blood was collected by venipuncture before and 1 month (28–42 days) after the concomitantly administered vaccines. Antibodies to diphtheria toxoid were determined using the micrometabolic inhibition test and reported as international units/mL (IU/mL). Antibodies to tetanus toxoid and pertussis antigens [pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin (PRN) and fimbriae types 2 and 3 (FIM)] were assessed by enzyme-linked immunosorbent assay (ELISA); tetanus antibodies were reported as IU/mL and pertussis antigens as ELISA units/mL (EU/mL). Antibodies to poliovirus antigens were measured by a Vero cell neutralization assay and reported as reciprocal dilution. Antibody to PRP was assessed by a Farr-type radioimmunoassay and reported as µg/mL. Antibody to HBsAg was measured using an enhanced chemiluminescence sandwich ELISA and reported as mIU/mL. Response to PCV7 was determined by measurement of antibodies to pneumococcal polysaccharides specific for serotypes 4, 6B, 9V, 14, 18C, 19F and 23F by ELISA and reported as µg/mL. Testing for antibodies to diphtheria, tetanus, pertussis and poliovirus antigens was performed by Sanofi Pasteur (Swiftwater, PA); testing for antibodies to PRP, HBsAg and pneumococcal antigens was performed by Merck Research Laboratories (West Point, PA).
The primary hypothesis in this study was that the investigational vaccine would induce a satisfactory antibody response to all its constituent antigens for Group A. The antibody responses were considered satisfactory if the lower limits of the 2-sided 95% confidence interval (CI) were greater than the predetermined limits for each antigen.
The targeted responses were based on previous knowledge and study results and the lower limits were 13–15% less than the targeted responses, representing clinical judgment as to satisfactory antibody responses. For pertussis antigens, seroconversion was defined as a 4-fold or greater antibody rise pre- to post-toddler dose. The targeted seroconversion rates for Group A were 90% (lower bound 75%) for PT antibody, 82% (lower bound 67%) for FHA antibody, 88% (lower bound 73%) for FIM antibody and 75% (lower bound 60%) for PRN antibody. For Hib, the targeted seroprotection response was 85% (lower bound 70%) of participants with a PRP antibody level >1 µg/mL. For hepatitis B, the targeted seroprotection rate was 95% (lower bound 80%) with an anti-HBs level ≥10 mIU/mL; for diphtheria, 95% (lower bound 80%) with a diphtheria antibody level ≥0.1 IU/mL; for tetanus, 98% (lower bound 85%) with a tetanus antibody level ≥0.1 IU/mL; and for poliovirus 98% (lower bound 85%) with antipoliovirus type 1, 2 and 3 titers ≥8 reciprocal dilution. The targeted responses were 2% higher for each of the vaccine antigens for Group B. Secondary immunogenicity outcomes included geometric mean antibody titers and 95% CI postvaccination as well as seroprotection/seroconversion rates for all antigens for Groups B and C. Safety outcomes included proportion of participants (95% CI) reporting solicited adverse events for 7 days postvaccination, unsolicited adverse events for 30 days postvaccination and serious adverse events anytime during the study. Immunogenicity analyses were performed using the intent-to-treat for immunogenicity population defined as participants that received the toddler dose, contributed at least 1 postvaccination blood and had a valid serology result for at least 1 antigen. The safety analysis was performed on the intent-to-treat population defined as all participants who received at least 1 dose of vaccine and who contributed at least 1 postvaccination safety measurement.
The sample size was calculated using the primary hypothesis criteria for the primary immunogenicity outcomes. A sample size of 135 evaluable participants for Group A with an estimated overall power of 91.1% (power of the individual immunogenicity endpoints ≥95.7%) was calculated to be able to detect a difference in antibody response of 13–15%. Assuming a 15% nonevaluable rate (including drop-outs), an enrollment sample size of 159 was selected. The Group B sample size was the same as that for Group A and would achieve an overall power of 92.3% (power of the individual immunogenicity endpoints ≥96%).
The fourth (toddler) doses of DTaP5, IPV, Hib, HepB and PCV7 were administered between September 2007 and April 2008 at 8 sites in Canada. Out of the 460 infants initially enrolled in the infant primary series study, 10 participants did not complete the primary series and 12 participants discontinued from the study between the infant series and the toddler dose (2 due to other adverse events, 4 lost to follow up and 6 due to voluntary withdrawal). A total of 438 (95.2%) continued with the toddler dose [Group A: 149 (94.9%); Group B: 145 (96.7%); Group C: 144 (94.1%)]. All but 1 participant in Group A, 1 participant in Group B and 2 participants in Group C provided safety data and formed the intention-to-treat population for the safety analysis. The intent-to-treat population for the fourth dose immunogenicity analysis comprised 148 participants in Group A, 140 participants in Group B and 139 participants in Group C. Reasons for exclusion from the analyses are depicted in Figure 1. The gender distribution of groups was similar at the time of the fourth dose, with 52.0% females in Group A, 42.9% in Group B and 54.7% in Group C. The mean age at the time of the toddler dose was 15.3 months (range 13.9–18.3) in Group A, 15.2 months range (14.6–18.4) in Group B and 15.1 months (14.4–16.8) in Group C. Many participants (79.1% in Group A, 81.4% in Group B and 82.7% in Group C) were Caucasian.
Rates of solicited adverse events were similar across the 3 vaccine groups. Erythema and tenderness were reported more frequently than swelling in the 3 groups during the 7 days postvaccination (Fig. 2A). Tenderness was reported by 48% (95% CI: 39.7–56.3) of Group A participants, 37.9% (30.0–46.4) of Group B participants and 38.7% (30.7–47.3) of Group C participants. Severe tenderness was reported by 3.4% (1.1–7.7), 1.4% (0.2–4.9) and 2.8% (0.8–7.1), respectively. Erythema was reported by 39.3–41.2% and swelling by 21.8–28.4% of the 3 vaccine groups; severe erythema and severe swelling was reported by 0–5.5% and 0–2.8%, respectively.
Irritability was the most common solicited systemic adverse event reported by 41.5–53.7% of participants of the 3 vaccine groups (Fig. 2B). Severe irritability was reported by only 0–2.7% of participants of the 3 vaccine groups. Decreased appetite (26.1–31.7%), drowsiness (19.7–25.5%) and crying (26.1–30.2%) were all commonly reported; severe systemic adverse events were all reported by <3.5% of participants in all vaccine groups. Fever (temperature ≥38°C) postvaccination was reported with similar frequency by participants in all groups ranging from 28.2–38.5% and severe fever (temperature >39.5°C) was reported by 0–4.1% of participants of the 3 vaccine groups.
Unsolicited adverse events were reported by 3.2% (1.0–7.3) of Group A participants, 4.0% (1.5–8.5) of Group B participants and 5.9% (2.7–10.9%) of Group C participants. Gastrointestinal disorders and upper respiratory infections were the most common unsolicited adverse events reported. Only 3 serious adverse events were reported: 2 cases in Group A, gastroenteritis 19 days postvaccination and bronchospasm 15 days postvaccination, and 1 case in Group C, febrile seizure 4 days postvaccination (concomitant diagnosis of roseola). None of the serious adverse events were deemed by the investigator to be related to vaccination.
Satisfactory antibody responses according to predetermined criteria were demonstrated for all antigens in Groups A and B. In Group C for which no criteria were set, the proportion of participants achieving a 4-fold response to FHA (55.2%; 95% CI: 46.4–63.8) was below the criteria set for Groups A and B (Table 2); this lower response to FHA was the result of both higher pre-fourth-dose and lower post-fourth-dose antibody titers. Although meeting the criteria set for Groups A and B, the proportion of participants in Group C with a 4-fold or greater antibody response to FIM (88%; 81.2–93) was also lower than participants in Group A (99.3%; 96.2–100). Post-toddler-dose seroprotection was demonstrated for PRP, HBsAg, diphtheria, tetanus and polioviruses in >95% of all participants.
Geometric mean antibody titers against pertussis antigens were similar in Groups A and B but were lower for PT, FHA and FIM in Group C (Table 3). Group C participants also had lower postvaccination antibody levels against tetanus and poliovirus type 2 compared with Groups A and B. In contrast, Group C achieved higher postvaccination antibody levels against PRP. Groups A and B had similar antibody levels against all antigens postvaccination except for diphtheria where Group B achieved higher antibody levels than Groups A and C. Antipneumococcal antibody levels were similar between Groups A and C who received the concurrent pneumococcal vaccine.
This study reports the results of the fourth (toddler) dose of the investigational hexavalent DTaP5-IPV-Hib-HepB in 15-month-old toddlers who had previously received 3 priming doses of the vaccine at 2, 4 and 6 months of age. Group A participants who received DTaP5-IPV-Hib-HepB and PCV7 at the same visit met the prespecified thresholds for acceptable immunogenicity and had similar reactogenicity and immunogenicity as Group B participants who received DTaP5-IPV-Hib-HepB and PCV7 at separate visits. Although adverse event profiles were similar, antibody responses in participants in Groups A and B were higher against pertussis antigens FHA, FIM and PT as well as against tetanus and poliovirus type 2 and lower against Hib PRP than in Group C participants who received separate injections at the same visit of the marketed, pentavalent DTaP5-IPV/Hib, PCV7 and HepB vaccines. Although Group C participants pre-toddler-dose had significantly lower anti-PRP antibody levels compared with Group B, post-toddler-dose levels were the higher in Group C than in Group A or B recipients. All 3 groups had substantial increases post-toddler-dose and achieved high levels of anti-PRP antibody. The safety and immunogenicity results of this fourth (toddler) dose differ in some ways from the results in these same participants after the primary series.11 After the primary series, participants in Groups A and B exceeded the predetermined threshold immunogenicity criteria for all antigens except for FHA, and antibody responses to diphtheria were significantly lower when PCV7 was administered concurrently at the same visit. Similar to this study, most of the antibody responses after the primary series were better in Groups A and Group B recipients of DTaP5-IPV-Hib-HepB than in Group C recipients, who were given the currently available pentavalent DTaP5-IPV/Hib, PCV7 and HepB vaccines separately.
As with the primary series,11 geometric mean antidiphtheria antibody levels were higher in participants who received the PCV7 vaccine on separate visits (Group B) than in participants who received both vaccines concurrently at the same visit (Groups A and C). This might be due to immune interference between the diphtheria protein cross-reactive material 197 used as the carrier protein in PCV7 and similar antigens contained in the pentavalent and hexavalent combination vaccines.11–13 Alternatively, the antidiphtheria antibody responses for Group B may have been boosted by the separate, additional administrations of the diphtheria protein cross-reactive material 197 of PCV7. In contrast to the primary series where Groups A and B did not meet the prespecified criterion for FHA (but Group C did), after the fourth (toddler) dose, both Groups A and B met the criteria for FHA whereas the response for FHA in Group C was lower.
The development of combination vaccines provides multiple benefits but there is risk of immune interference among the antigen components as well as with concurrently administered vaccines.12,13 PCV7 (or now PCV13) is given at the same visit as the combination vaccines; this study demonstrated that the antibody response to PCV7 was similar when given concurrently with the hexavalent DTaP5-IPV-Hib-HepB vaccine as when given with the currently licensed pentavalent DTaP5-IPV/Hib vaccine. The inclusion of HepB in the hexavalent vaccine did not have any detrimental effect on most of the other antigens included in the combination product.14,15 Indeed, antibody levels were higher against PT, FHA, FIM, tetanus and poliovirus type 2 in recipients of the hexavalent product. Only anti-Hib PRP antibodies were lower in DTaP5-IPV-Hib-HepB recipients compared with DTaP5-IPV/Hib recipients. Interestingly, lower anti-Hib PRP antibodies16–18 and anti-FHA antibodies19 have also been reported after another manufacturer’s hexavalent DTaP-IPV-Hib-HepB vaccine compared with their pentavalent DTaP-IPV/Hib product.
While this study was successful in achieving its objectives, its design has several limitations. Although the primary immunological outcomes were objective laboratory measures, the study was open label. In order to provide a fully double- blinded design, we would have had to administer a placebo injection to participants in Group B at the first study visit (when Group A and C received PCV7) and a placebo injection to participants in Groups A and C at the second visit (when Group B received PCV7). While this would have ensured full blinding, the use of placebo injections in infants and toddlers is problematic, particularly where the primary serological outcomes can be measured objectively. The study was also not powered to demonstrate noninferiority between the 3 vaccine regimens but rather to demonstrate satisfactory antibody responses based on thresholds that were predetermined from prior immunogenicity studies. A larger study powered for noninferiority would have enabled us to make direct comparisons between the vaccine groups. Finally, while this study demonstrated that administration of PCV7 concurrently with the combination vaccine had no adverse effect on the antibody responses to the combination vaccine (future studies will assess concurrent administration of PCV13 vaccine), antipneumococcal antibody levels were not measured 1 month postvaccination in Group B so we are unable to comment on the effect of separate or concurrent administration of the combination vaccines on the antibody response to the PCV serotypes. Similarly, we did not measure antibodies against HBsAg in Group C recipients, who received HepB as a separate injection.
The development of this investigational hexavalent DTaP5-IPV-Hib-HepB vaccine will provide another option for the vaccination of infants and toddlers against these 6 antigens. While another manufacturer’s product (Infanrix-Hexa, GlaxoSmithKline, Rixensart, Belgium) is already on the market and widely used in some jurisdictions, a second product would be beneficial for ensuring adequate global supply and potentially decreasing vaccine costs. This investigational hexavalent DTaP5-IPV-Hib-HepB has now undergone phase 1 and 2 studies to optimize the formulation20–22 and has been demonstrated to be well-tolerated and immunogenic when given concurrently with PCV7 in infants11 and for the fourth (booster) dose in toddlers. The favorable results of the current study support the continued development of the vaccine.
This study was funded by Sanofi Pasteur and Merck. The authors thank the study investigators and their staff at the participating trial centers for their careful attention to detail and the children and their families for participating in the study. We thank the serology groups of Sanofi Pasteur and Merck for serology testing. The authors thank Robert Lersch of Sanofi Pasteur and Julia Gage of Gage Medical Writing who produced the tables and graphs and coordinated the article revision process.
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