This was a phase 3, parallel-group, randomized, active controlled, double-blind, multicenter trial carried out in four Pediatric Departments (Milan, Novara, Rome, Bologna) and five Public Health Departments (Ragusa, Sassari, Genoa, Massafra, Palermo) in Italy. Approval was obtained from the respective Ethics Committees of the participating study sites. Written informed consent for participation in the study was obtained from the subjects' parents/guardians. Subjects were randomly assigned in a 1:1 ratio to one of the two vaccine groups to receive either PCV13 plus DTaP-HBV-IPV/Hib vaccine or PCV7 plus DTaP-HBV-IPV/Hib vaccine at 3, 5, and 11 months of age. The randomization schedule was executed by Wyeth's Clinical Operations Randomization Environment system, which is accessible via the internet and by telephone 24 h a day. Treatment allocation was concealed from all subjects, study staff, and those assessing the outcomes.
At approximately 3, 5, and 11 months of age, subjects received a single intramuscular injection of pneumococcal conjugate vaccine (either PCV7 or PCV13) into the left thigh and a single intramuscular injection of DTaP-HBV-IPV/Hib vaccine into the right thigh. Both vaccines were administered with a 22-gauge, 2.5-cm-long needle. A blood sample was collected at approximately 6 months of age or 1 month after the second dose of the primary series. A second blood sample was drawn immediately before the third vaccination (toddler dose), and a third blood sample was drawn 1 month after the toddler dose.
Subjects eligible to participate in this study were healthy infants, as determined by medical history and physical examination. They were to be 3 months of age (75 to 105 days) at the time of enrollment, to be available for the entire study period, and to be born at more than 32 weeks of gestational age with a birth weight >2,000 g. In addition, the subjects' parents/guardians had to be available to be contacted by telephone and able to complete all of the relevant study procedures.
Excluded were subjects with any of the following: previous vaccination with a licensed or investigational pneumococcal conjugate vaccine or Hib conjugate, diphtheria, tetanus, pertussis, polio, or hepatitis B vaccine; bleeding diathesis or conditions associated with a prolonged bleeding time that would contraindicate intramuscular injection; known or suspected immune deficiency or suppression; a history of culture-proven Streptococcus pneumoniae or H. influenzae type b invasive disease; major known congenital malformation or serious chronic disorder; or a history of seizures, including febrile seizures, or a significant stable or evolving neurological disorder. Also excluded were children who had received blood products or gamma globulin, who were participating in another investigational study, or who were direct descendants of the study site personnel.
A complete medical history of each child was obtained and a complete physical examination was performed at visit 1, before randomization and administration of any study vaccinations.
PCV7 was equivalent to licensed Prevenar (Wyeth Pharmaceuticals Inc., Philadelphia, PA). PCV13 was formulated in a manner similar to that used for PCV7. It contains serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F, as well as serotypes 1, 3, 5, 6A, 7F, and 19A. As with PCV7, each of the polysaccharides is covalently conjugated to a common carrier protein, CRM197, a nontoxic variant of diphtheria toxin. PCV13 contains 2.2 μg of each polysaccharide, except for 4.4 μg of 6B, in 5.0 mM succinate buffer with 0.125 mg of aluminum as aluminum phosphate per 0.5-ml dose. The presentations of PCV13 and PCV7 used identical prefilled syringes.
DTaP-HBV-IPV/Hib vaccine was the commercially available Infanrix hexa (GlaxoSmithKline Biologicals, Rixensart, Belgium). A 0.5-ml dose of the vaccine was prepared for use according to the vaccine's labeling.
Subjects were observed for 30 min after each vaccination for any immediate reaction. Any AE noted was recorded at this time. After discharge from the vaccination center, local reactions (redness, swelling, and tenderness), systemic events (decreased appetite, irritability, increased or decreased sleep), rectal temperature, and use of antipyretic medication to treat or prevent symptoms were monitored by the parents/guardians for 4 days, including the day of vaccination (day 1), and recorded in an electronic diary (e-diary). Parents/guardians measured the diameter of redness or swelling with a caliper provided for the purpose and recorded the measurement (1 to 14 or 14+) in whole-number increments in the e-diary. Each caliper unit represented 0.5 cm. Tenderness was recorded as none, present, or interfering with limb movement. Rectal temperature was recorded every evening and at any other time during the 4 days a fever was suspected. The highest temperature for each day was recorded in the e-diary.
Other systemic events (decreased appetite, irritability, increased sleep, and decreased sleep), and the use of antipyretic medications to prevent or treat symptoms were also recorded in the e-diary by the parents/guardians for 4 days after each vaccination. AEs (defined as any untoward, undesired, or unplanned signs, symptoms, diseases, or lab test results) were also recorded.
Antibody responses to hepatitis B, Hib antigen polyribosylribitol phosphate (PRP), and pertussis antigens (pertussis toxoid [PT], filamentous hemagglutinin [FHA], and pertactin [PRN]), tetanus toxoid, diphtheria toxoid, and poliovirus (Sabin strains 1, 2, and 3) were measured in the blood drawn 1 month after the primary immunization and 1 month after the toddler dose in all subjects with available sera. Standard enzyme-linked immunosorbent assay (ELISA) techniques were used to measure concentrations of antibodies against tetanus toxoid, PRP, hepatitis B virus, PT, FHA, and PRN (30
). Antibodies to poliovirus Sabin strains 1, 2, and 3 were evaluated with a poliovirus in vitro
plaque neutralization assay (31
), and diphtheria toxoid antibodies were evaluated by an anti-diphtheria toxoid ELISA (25
Serum concentrations of anti-capsular polysaccharide IgG for each of the 13 pneumococcal serotypes were determined for all subjects in the PCV13 group for the 1 month post primary immunization and pre-toddler dose serum samples. They were also determined in all subjects for samples drawn 1 month after the toddler dose. The assay used was a standardized antipolysaccharide ELISA as previously described (6
Serum opsonophagocytic activity induced by PCV13 was measured using previously described methods (17
) on sera obtained 1 month after the primary series and 1 month after the toddler dose in a randomly selected subset of approximately 100 subjects in the PCV13 group.
All of the immunological assays were performed in Wyeth laboratories.
Sample size was estimated on the basis of the proportion of immunogenicity responders (see below) in each vaccine group. Data from two earlier Wyeth studies on pneumococcal vaccine and a European Agency for the Evaluation of Medicinal Products scientific discussion document for Infanrix hexa were used in the estimation. A sample size of 85 evaluable subjects per group provided at least 99% power to declare noninferiority of the hepatitis B antigen when administered with PCV13 relative to the hepatitis B antigen administered with PCV7. A sample size of 225 evaluable subjects per group at the toddler dose would provide a 70% power to declare noninferiority for all 13 pneumococcal antigens using a noninferiority criterion of 0.10 (lowest limit of the 95% confidence interval [CI] for the difference between the proportions of responders in the PCV13 and PCV7 groups greater than or equal to −0.1) and a two-sided type 1 error of 0.05. Assuming a dropout rate of, at most, 25%, 600 subjects overall were to be enrolled to ensure that 450 subjects were evaluable at the toddler dose.
Inclusion criteria and statistical analysis.
The primary objective was to demonstrate noninferiority of the immune response to hepatitis B virus after the toddler dose. Immune responses to all other concomitant vaccine antigens and to PCV13 and PCV7 were also evaluated. The safety profile of PCV13, as measured by the incidence of local reactions, systemic events, and AEs, was also evaluated. The evaluable population in the study consisted of all vaccinated subjects who met all of the eligibility criteria, complied with protocol defined criteria, had follow-up blood drawn 27 to 56 days postvaccination, and had available immunization data for at least one pneumococcal serotype and one DTaP-HBV-IPV/Hib antigen. A separate evaluable toddler population consisted of all subjects who completed the primary immunization on schedule, were immunized at the appropriate age for the toddler dose, had blood samples drawn on schedule (time between toddler dose and blood draw, 27 to 56 days; predose blood drawn <7 days before dose administration), and had available postvaccination data for at least one pneumococcal serotype and for at least one of the DTaP-HBV-IPV/Hib antigens. In both randomized vaccine groups, two subgroups were defined, subjects with at least one valid assay result for a concomitant vaccine antigen and those subjects with at least one valid assay result measuring the immune response to pneumococcal vaccine. Rates of local reactions and systemic events over the study period were calculated for each treatment group, and differences in the incidence of local reactions and systemic events in the PCV13 group relative to the incidence rates in the PCV7 group were reported. Fever was categorized as mild if the rectal temperature was ≥38.0°C but ≤39.0°C, moderate if it was >39.0°C but ≤40.0°C, and severe if it was >40.0°C. Redness and swelling were categorized as absent, mild (0.5 to 2.0 cm), moderate (2.5 to 7.0 cm), or severe (>7.0 cm). The safety population included all subjects who received at least one dose of the study vaccine. Separate populations were defined for each dose, and a two-sided Fisher exact test was used to perform all of the comparisons.
For the concomitantly administered vaccinations, the primary immunological comparison was the hepatitis B antigen immune response in subjects receiving PCV13 relative to the hepatitis B antigen immune response in subjects receiving PCV7. The primary endpoint for each antigen was the proportion of the subjects who achieved a prespecified antibody concentration as follows: hepatitis B antibody level, ≥10 milli-IU/ml; pertussis vaccine (PT, FHA, and PRN), ≥5 EU (ELISA units)/ml; Hib antigen (PRP) antibody level, ≥0.15 μg/ml; diphtheria toxoid antibody level, ≥0.1 IU/ml; tetanus toxoid antibody level, ≥0.1 IU/ml; poliovirus neutralizing antibody titer, >1:8. Alternate levels of ≥7.82 EU/ml for FHA and ≥1.0 μg/ml for PRP were also prespecified. An additional comparison for pertussis compared responders at the antibody level achieved by 95% of the subjects in the PCV7 group; the level was determined for each antigen (pertussis vaccine PT, FHA, and PRN) separately. For concomitant antigens, 95% CIs for the proportion of responders and for the difference in proportions (PCV13 − PCV7 reference) were calculated, with noninferiority declared if the lower bound of the two-sided 95% CI for the difference in proportions was greater than −10%.
The primary immunogenicity endpoints for each of the pneumococcal serotypes were the proportion of the subjects in the PCV13 group attaining serotype-specific antipolysaccharide IgG concentrations of ≥0.35 μg/ml 1 month after the infant series. As recommended by the WHO, this antibody concentration serves as a reference value for the comparison of novel pneumococcal conjugate vaccines with PCV7 to assess the potential for efficacy against invasive disease (44
). The geometric mean IgG antibody concentration measured 1 month after the primary series also served as a primary endpoint. The pneumococcal IgG immune responses induced by the PCV13 relative to those of PCV7 1 month after the toddler dose were evaluated as secondary endpoints. Within each vaccine group and for each pneumococcal serotype, the proportion of the subjects with a specific IgG antibody concentration of ≥0.35 μg/ml and 95% CIs on the proportion were calculated. To assess treatment differences, 95% CIs on the difference in proportions (PCV13 − PCV7 reference) were calculated. For each serotype, noninferiority was declared if the lower bound of the two-sided 95% CI for the difference in proportions was greater than −0.10. For the pneumococcal opsonophagocytic assay (OPA) titers, the proportions of the subjects with OPA titers of at least 1:8 were calculated and analyzed in the same way as for IgG responders. According to WHO guidelines, an OPA response above a titer of 1:8 correlates with efficacy for some serotypes (11
Within each vaccine group and for each antibody concentration or OPA titer, geometric means were also calculated. Each concentration/titer was logarithmically transformed for the analysis. Two-sided 95% CIs were constructed by back transformation of the CIs for the mean of logarithmically transformed assay results computed using the Student t distribution. Rises (n-fold) in antibody concentrations from before to after the toddler dose were summarized by geometric means and CIs and computed using the logarithmically transformed assay results. To assess differences between the two vaccine groups, two-sided 95% CIs for the ratio of the geometric mean concentrations (GMCs; PCV13/PCV7 reference) were constructed. The noninferiority criterion was met if the lower limit of the 95% CI of the ratio was >0.5 (2-fold criterion). In addition, for the PCV13 group, the ratio of the geometric mean n-fold rises (GMFRs) in post- to pretoddler GMCs and the corresponding two-sided 95% CIs were calculated.