Immunogenicity and Safety of a Combination Diphtheria, Tetanus Toxoid, Acellular Pertussis, Hepatitis B, and Inactivated Poliovirus Vaccine Coadministered with a 7-Valent Pneumococcal Conjugate Vaccine and a Haemophilus Influenzae Type b Conjugate Vaccine

Subjects 

The study was conducted at 22 sites in the United States, all of which received approval by their institutional review board before initiation. Healthy infants 2 months (6 to 12 weeks) of age at the time of first vaccination were enrolled after written informed consent was obtained from their parents or guardians. Infants were excluded if any of the following was present: premature birth (<36 weeks’ gestation); previous vaccination against or history of diphtheria, tetanus, pertussis, poliomyelitis, H influenzae type b, or S pneumoniae disease, or hepatitis B disease or more than 1 previous dose of HepB; confirmed or suspected immune dysfunction; previous chronic administration of immunosuppressants or other immune-modifying drugs; history of hypersensitivity to any components of the vaccines; major congenital defects or serious chronic illness; neurologic or seizure disorder; or no telephone access.

Vaccines 

Six different vaccines were used in this study. Each 0.5-mL dose of the DTaP-HepB-IPV vaccine (PEDIARIX) was supplied in a prefilled syringe from a single lot and was formulated to contain 25 Lf (limit of flocculation unit) diphtheria toxoid, 10 Lf tetanus toxoid, 25 μg pertussis toxoid, 25 μg filamentous hemagglutinin, 8 μg pertactin, 10 μg hepatitis B surface antigen (recombinant), 40 D-antigen units of type 1 (Mahoney), 8 D-antigen units of type 2 (MEF-1), and 32 D-antigen units of type 3 (Saukett) of polioviruses. The DTaP vaccine (Infanrix, GlaxoSmithKline Biologicals, Rixensart, Belgium) contained 25 Lf diphtheria toxoid, 10 Lf tetanus toxoid, 25 μg pertussis toxoid, 25 μg filamentous hemagglutinin, and 8 μg pertactin per 0.5-mL dose in a single-dose vial. The hepatitis B vaccine (Engerix-B, GlaxoSmithKline Biologicals, Rixensart, Belgium) contained 10 μg hepatitis B surface antigen (recombinant) per 0.5-mL dose in a prefilled syringe. Each 0.5-mL dose of the commercially available PCV-7 vaccine (Prevnar) contained 2 μg of each of the pneumococcal polysaccharide serotypes 4, 9V, 14, 18C, 19F, and 23F, and 4 μg of 6B coupled to the CRM197 carrier protein and was supplied in a single-dose vial. The Hib vaccine (HibTITER, Wyeth Pharmaceuticals, Inc, Philadelphia, Pa) contained 10 μg of Haemophilus b saccharide conjugated to CRM197 protein per 0.5-mL dose in a single-dose vial. The commercially available IPV vaccine (IPOL, Sanofi Pasteur SA, Lyon, France) contained 40 D-antigen units of type 1 (Mahoney), 8 D-antigen units of type 2 (MEF-1), and 32 D-antigen units of type 3 (Saukett) of poliovirus per 0.5-mL dose in a prefilled syringe.

Study Design 

In this open-label study, infants eligible for inclusion were randomly assigned to one of three study groups, according to an Internet randomization system, with 10% randomness and the center as minimization factor. This is a classic randomization method of minimization in that it corrects the imbalance between treatments within a center in 90% of the assignments (ie, with 90% probability) and allocates randomly to one of the three groups in 10% of the assignments (ie, with 10% probability). The vaccination schedule for the study groups is presented in the Figure. Intramuscular injections of DTaP-HepB-IPV or DTaP vaccine were administered in the upper right anterolateral thigh; HepB vaccine was administered in the lower right anterolateral thigh; and PCV-7 and Hib vaccines were administered in the upper and lower left anterolateral thigh, respectively. When more than one vaccine was administered into the same leg, the injections were separated by at least 2 inches. IPV vaccine was administered by subcutaneous injection in the left deltoid.

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Figure. Study design. *For the separate administration group, HepB was not to be administered at 4 months of age to subjects who received a dose of hepatitis B vaccine before enrollment in this study. DTaP indicates diphtheria–tetanus–acellular pertussis vaccine; HepB, hepatitis B vaccine; Hib, H influenzae type b vaccine; IPV, inactivated poliovirus vaccine; mos, months; PCV-7, pneumococcal conjugate vaccine; wks = weeks.

Blood samples were collected from all subjects at 2 months of age (before the first vaccination) and 7 months of age (1 month after the third DTaP-HepB-IPV or DTaP vaccination).

Serologic Evaluations 

Anti-S pneumoniae antibody testing only was performed in a blinded fashion at the GlaxoSmithKline Laboratory in Rixensart, Belgium; all other assays were performed at the University of Rochester, Rochester, NY (MEP Laboratories). Serum samples were tested for the presence of antibodies to all vaccine antigens. Standardized enzyme-linked immunosorbent assays (ELISAs) were used to assess antibody concentrations to diphtheria and tetanus toxoids, pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin (PRN), polyribosylribitol (PRP), and 7 S pneumoniae serotype-specific antibodies (to 4, 6B, 9V, 14, 18C, 19F, and 23F). The cut-off of the ELISA for diphtheria and tetanus toxoids was 0.1 IU/mL. A concentration of 0.1 IU/mL or above was considered seropositive and seroprotective. The cut-off of the ELISA for PT, FHA, and PRN were 5 ELISA units (EL.U)/mL. Vaccine response for PT, FHA, and PRN was defined as the appearance of antibodies in infants who were initially seronegative (ie, with concentrations less than the cut-off value) or at least maintenance of prevaccination antibody concentrations in subjects who were initially seropositive (ie, with concentrations greater than or equal to the cut-off value). Antibody concentrations for hepatitis B surface antigen (anti-HBsAg) were measured by ELISA, using a commercially available kit (AUSAB, Abbott Laboratories, Abbott Park, Ill), with the cut-off at 10 mIU/mL. Concentrations greater than or equal to this value were considered to be seroprotective. For PRP, the cut-off of the ELISA test was 0.15 μg/mL. Concentrations of 0.15 μg/mL or above and 1.0 μg/mL or above were analyzed. Neutralizing antibodies against poliovirus types 1, 2, and 3 were determined by a microneutralization assay and were expressed as dilution titers. Results were expressed as the reciprocal of the highest dilution of serum showing 50% virus neutralization. Antibody titers of 1:8 or higher were considered seropositive and seroprotective. S pneumoniae serotype-specific antibody concentrations were determined by comparing the ELISA curves with a standard reference serum, with the cut-off at 0.05 μg/mL. Concentrations greater than or equal to this value were considered seropositive.

Safety Evaluations 

All infants were observed for 30 minutes after each vaccination. Parents were asked to record temperatures, other general symptoms (irritability/fussiness, loss of appetite, and drowsiness), local symptoms (pain, redness, and swelling at the injection sites), and concomitant medications on the day of vaccination (day 0) and for the next 3 days (days 1 through 3). They were supplied with a diary to assist them in reporting symptoms, a gauge for measuring reactions (redness and swelling) at the injection sites, and a thermometer to record rectal temperatures. Unsolicited adverse events were recorded for the entire study period. For each symptom or event reported, parents were asked if they sought medical advice (defined as a visit with medical personnel). During the extended safety follow-up period (6 months after the last dose of study vaccine), serious adverse events, onset of chronic illness (eg, diabetes, autoimmune disease, asthma, and allergies), and emergency department visits and physician office visits not related to well-child care, vaccination, or common acute illnesses (eg, upper respiratory tract infections, otitis media, pharyngitis, and gastroenteritis) were reported.

Statistical Analysis 

Primary immunogenicity analyses were based on the according-to-protocol cohort, which included all subjects who met the eligibility criteria, who complied with the procedures of the protocol, and for whom assay results were available for at least one study vaccine antigen 1 month after the three-dose primary vaccination series.

Geometric mean antibody concentrations (GMCs) or geometric mean antibody titers (GMTs) and seropositivity or seroprotection rates were calculated with their 95% confidence intervals (CIs) for each group at each blood-sampling time point. One month after the third dose, differences between the Combination Vaccine Group and each of the other two groups (Separate and Staggered Vaccine Groups) were computed with standardized asymptotic 95% CIs for pertussis vaccine response rates and seroprotection rates for diphtheria, tetanus, hepatitis B, poliovirus types 1, 2, and 3, and Hib.8 For each antigen, antibody GMC/GMT ratios (Separate Vaccine Group divided by Combination Vaccine Group, and Staggered Vaccine Group divided by Combination Vaccine Group) were computed with 90% and 95% CIs, using an analysis of covariance (ANCOVA) model on the logarithm10 transformation of the antibody concentrations/titers. The ANCOVA model included the vaccine group as fixed effect (all three groups) and the prevaccination concentration/titer as regressor.

The criteria for clinical noninferiority for the primary objective included 1) the upper limit of the 90% CI on the antibody GMC ratio (Separate Vaccine Group over Combination Vaccine Group) below 1.5 for each pertussis antigen; 2) the upper limit of the 95% CI on the absolute difference (Separate Vaccine Group minus Combination Vaccine Group) for seroprotection rate below 10% for diphtheria and tetanus antigens; and 3) the upper limit of the 95% CI on the absolute difference for seroprotection rate below 5% for the three poliovirus antigens. Noninferiority for hepatitis B was not assessed because HepB was not to be administered at 4 months of age to infants in the Separate Vaccine Group who received a dose of HepB before enrollment in the study. Additional exploratory analyses were performed to characterize group difference in immunogenicity between the combination vaccine group and each of the control groups: statistical difference was based on 0% being included in the 95% CI for group difference in seroprotection rate and on 1 being included in the 95% CI for group GMC ratio.

All safety analyses were descriptive and based on the modified intent-to-treat cohort, which included all vaccinated infants for whom safety data were available. Safety data were categorized, and analyses were performed overall, per subject, for the three-dose vaccination series. The percentage of infants having specific symptoms within the 4 days after vaccination were computed with the exact 95% CI, according to the type of symptom, intensity, and relation to vaccination.9 Differences between groups in percentage of subjects reporting solicited symptom(s) were evaluated through two-sided Fisher’s exact tests. A P value below .05 was used to highlight possible differences. Serious adverse events reported during the entire study, including the extended safety follow-up period, were described.

Study Population 

A total of 575 infants were enrolled and randomly assigned to the Separate Vaccine Group (n = 188), the Combination Vaccine Group (n = 199), and the Staggered Vaccine Group (n = 188). The modified intention-to-treat cohort for safety included 573 infants (188, 198, 187 in the Separate, Combination, and Staggered Vaccine Groups, respectively), and the according to protocol cohort for immunogenicity included 486 infants (156, 169, and 161 in the Separate, Combination, and Staggered Vaccine Groups, respectively). Among the 575 infants enrolled, the median age at first dose was 9 weeks, 51.5% were female, and 84% were Caucasian. The demographics were similar among the three groups.

During the course of the study, 43 infants were withdrawn (16 each from the Separate and Combination Vaccine Groups, and 11 from the Staggered Vaccine Group). The reasons for withdrawal were loss to follow-up (25 infants), withdrawal of consent (9 infants), moving from study area (7 infants), and protocol violation (2 infants). No subject was withdrawn because of an adverse event.

Immunogenicity 

Criteria for noninferiority were met for all primary end points. The upper limits of the two-sided 95% CI on the absolute difference (Separate Vaccine Group minus Combination Vaccine Group) in the seroprotection rates for diphtheria (2.12%), tetanus (0.32%), and poliovirus types 1, 2, and 3 (2.24% for each) were below the predefined clinical limits of noninferiority (10% for diphtheria and tetanus, and 5% for poliovirus) (Table I; available at www.jpeds.com). Likewise, the upper limits of the 90% CI on the ratios of the adjusted antibody GMCs (Separate Vaccine Group/Combination Vaccine Group) for PT (0.70), FHA (1.00), and PRN (0.99) were below the predefined clinical limit for noninferiority of 1.5 (data not shown). Seroprotection rates for diphtheria, tetanus, and poliovirus were high in the Combination and Separate Vaccine Groups (Table I). Seroprotection rates for hepatitis B did not differ between the two groups, regardless of hepatitis B vaccination status before study entry. There were no statistically significant differences between the two groups in seroprotection rates for PRP. For anti–S pneumoniae antibodies, seropositivity was high in all groups. Although the comparison between the Combination and the Staggered Vaccine Groups was not a primary objective, the criteria for noninferiority as defined for the primary end points also were met for the comparisons between these two groups.

Table I. Seroprotection, seropositivity, and vaccine response rates 1 month after third dose of DTaP or DTaP-HepB-IPV (according-to-protocol cohort for immunogenicity)

		Combination Vaccine Group (n = 154-168)		Separate Vaccine Group (n = 141-156)		Difference (Separate minus Combination)		Staggered Vaccine Group (n = 149-158)		Difference (Staggered minus Combination)
	End point	n	Rate (%)	n	Rate (%)	Diff in rates (%)	95% CI	n	Rate (%)	Diff in rates (%)	95% CI
	Anti-D ≥0.1 IU/mL	167	99.4	153	98.7	−0.70	−4.05,2.12‡	157	100.0	0.60	−1.80,3.29
	Anti-T ≥0.1 IU/mL	168	100.0	152	98.1	−1.94	−5.54,0.32‡	158	100.0	0.00	−2.37,2.24
	Anti-HBsAg ≥10 mIU/mL⁎†	164	98.2	152	98.7	0.50	−3.01,4.01	158	100.0	1.80	−0.60,5.15
	Vaccine response to PT	153	98.7	135	95.1	−3.64	−8.69,0.31	148	97.4	−1.34	−5.44,2.26
	Vaccine response to FHA	152	98.7	136	96.5	−2.25	−6.89,1.52	150	98.0	−0.66	−4.46,2.87
	Vaccine response to PRN	143	91.7	135	95.1	3.40	−2.50,9.46	145	94.2	2.49	−3.44,8.62
	Anti-poliovirus 1 ≥1:8	168	100.0	153	100.0	0.00	−2.45,2.24‡	156	100.0	0.00	−2.40,2.24
	Anti-poliovirus 2 ≥1:8	168	100.0	153	100.0	0.00	−2.45,2.24‡	156	100.0	0.00	−2.40,2.24
	Anti-poliovirus 3 ≥1:8	168	100.0	153	100.0	0.00	−2.45,2.24‡	156	100.0	0.00	−2.40,2.24
	Anti-PRP ≥0.15 μg/mL	168	100.0	154	99.4	−0.65	−3.56,1.60	158	100.0	0.00	−2.37,2.24
	Anti-PRP ≥1.0 μg/mL	161	95.8	141	91.0	−4.87	−10.91,0.55	150	94.9	−0.90	−6.02,3.97
	Anti–S pneumoniae serotype ≥0.05 μg/mL
	4	163	99.4	156	100.0			155	100.0
	6B	156	95.7	134	90.5			148	96.1
	9V	163	100.0	151	100.0			150	100.0
	14	166	100.0	156	100.0			154	100.0
	18C	166	99.4	153	100.0			156	100.0
	19F	160	99.4	147	100.0			149	100.0
	23F	158	97.5	141	96.6			150	98.0

D indicates diphtheria; DTaP, diphtheria–tetanus–acellular pertussis vaccine; FHA, filamentous hemagglutinin; HBsAg, hepatitis B surface antigen; HepB, hepatitis B vaccine; IPV, inactivated poliovirus vaccine; n, number of subjects with available results; PRN, pertactin; PRP, polyribosylribitol; PT, pertussis toxin; T, tetanus. Vaccine response to PT, FHA, and PRN is defined as the appearance of antibodies in subjects who were initially seronegative (ie, with concentrations less than the cut-off value) or at least maintenance of prevaccination antibody concentrations in subjects who were initially seropositive (ie, with concentrations greater than or equal to the cut-off value).

⁎ Regardless of hepatitis B vaccination status before study entry. † No clinical limit defined for noninferiority; the 95% CIs of group differences in rates will not be used to draw conclusions on noninferiority of Combination Group to Separate Group but rather to provide information on the comparability of the two groups. ‡ Noninferiority criterion met: Upper limit below the clinical limit for noninferiority.

For all three groups after the third dose, seroprotection rates ranged from 98.7% to 100% for diphtheria and from 98.1% to 100% for tetanus (Table I). For antidiphtheria, postvaccination antibody GMCs were significantly higher in the Staggered Vaccine Group compared with the Combination or Separate Vaccine Group (Table II). For antitetanus, postvaccination antibody GMCs were significantly lower in the Separate Vaccine Group compared with the Combination Vaccine Group (Table II). Postvaccination response rates among the three groups ranged from 91.7% to 98.7% for PT, FHA, and PRN (Table I). The postvaccination antibody GMC for PT was significantly lower in the Separate Vaccine Group compared with the Combination Vaccine Group (Table II).

Table II. Antibody GMCs or GMTs 1 month after third dose of DTaP or DTaP-HepB-IPV (according-to-protocol cohort for immunogenicity)

		Combination Vaccine Group (n = 138-168)		Separate Vaccine Group (n = 126-156)		Staggered Vaccine Group (n = 135-158)
	Antibody	GMC/GMT	95% CI	GMC/GMT	95% CI	GMC/GMT	95% CI
	Anti-D (IU/mL)	1.987	1.761,2.242	1.564†	1.354,1.806	3.238†	2.889,3.630
	Anti-T (IU/mL)	2.428	2.180,2.704	1.346†	1.166,1.554	2.240	2.017,2.487
	Anti-PT (EL.U/mL)	48.7	42.8,55.4	28.9†	25.2,33.2	50.3	44.6,56.7
	Anti-FHA (EL.U/mL)	113.7	101.5,127.3	96.4	85.5,108.7	117.7	105.9,130.9
	Anti-PRN (EL.U/mL)	93.7	82.3,106.7	79.1	66.8,93.6	99.5	87.5,113.2
	Anti-HBsAg (mIU/mL)⁎	1123.6	912.0,1384.2	667.5†	534.1,834.3	1515.3	1250.6,1835.9
	Anti-poliovirus 1 titer	678.0	568.1,809.2	225.0†	191.3,264.7	793.1	660.3,952.7
	Anti-poliovirus 2 titer	578.3	479.3,697.8	228.1†	198.1,262.7	608.8	500.8,740.2
	Anti-poliovirus 3 titer	1269.2	1061.3,1517.8	454.2†	390.6,528.2	1167.5	973.9,1399.6
	Anti-PRP (μg/mL)	9.619	7.995,11.572	9.246	7.378,11.587	8.384	7.02,10.013
	Anti–S pneumoniae Serotype (μg/mL)
	4	1.74	1.54,1.98	2.07	1.81,2.37	1.62	1.44,1.83
	6B	0.80	0.65,0.99	0.67	0.52,0.87	0.59	0.49,0.72
	9V	1.55	1.36,1.77	1.60	1.39,1.85	1.11†	0.97,1.28
	14	4.68	4.04,5.43	6.32	5.39,7.41	4.51	3.91,5.19
	18C	2.63	2.31,3.00	2.96	2.53,3.47	2.37	2.06,2.72
	19F	1.09	0.95,1.25	1.05	0.91,1.22	0.75†	0.66,0.86
	23F	1.48	1.23,1.79	1.81	1.45,2.25	1.29	1.09,1.53

D indicates diphtheria; DTaP, diphtheria–tetanus–acellular pertussis vaccine; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; GMT, geometric mean titer; HBsAg, hepatitis B surface antigen; HepB, hepatitis B vaccine; IPV, inactivated poliovirus vaccine; n, number of subjects; PRN, pertactin; PRP, polyribosylribitol; PT, pertussis toxin; T, tetanus.

⁎ Total infants regardless of hepatitis B vaccination status before entry. † Statistically significant difference between the Combination Vaccine Group and either the Separate or Staggered Vaccine Group.

The percentage of infants who received a dose of HepB vaccine before study entry was 23%, 21%, and 25% in the Combination, Separate, and Staggered Vaccine Groups, respectively. In the Separate Vaccine Group, however, infants who received a dose of HepB vaccine before enrollment did not receive it at 4 months of age and therefore only received three doses overall. As a result, postvaccination HepB antibody GMCs were significantly lower in the Separate Vaccine Group than in the Combination Vaccine Group (Table II); hepatitis B seroprotection rates did not differ among the three groups (98.2% to 100%) (Table I).

After the third dose in all groups, seroprotection rates for all three types of poliovirus were 100% (Table I). Postvaccination antibody GMTs for the three types of poliovirus were significantly lower in the Separate Vaccine Group than in the Combination Vaccine Group (Table II). More than 99.4% of infants in all groups had anti-PRP antibody concentrations of 0.15 μg/mL or higher, and more than 91.0% had anti-PRP concentrations of 1.0 μg/mL or above after the third dose (Table I). Postvaccination GMCs for anti-PRP were similar among the groups (Table II).

With the exception of serotypes 9V and 19F, the antibody GMCs of the 7 S pneumoniae serotypes did not differ among the Combination Vaccine Group and the Separate and Staggered Vaccine Groups (Table II). The antibody GMCs of serotypes 9V and 19F were significantly lower in the Staggered Vaccine Group compared with the Combination Vaccine Group.

Safety 

General Symptoms 

Overall, irritability was the most frequently reported general symptom in all groups and was significantly higher in the Combination Vaccine Group compared with the Separate or Staggered Vaccine Groups (P = .002 and P = .005, respectively) (Table III; available at www.jpeds.com). Incidence rates of any drowsiness, irritability, and loss of appetite were significantly higher in the Combination Vaccine Group compared with the Staggered Vaccine Group (P < .05).

Table III. Overall incidence of solicited general and local symptoms reported within 4 days after vaccination in the per subject analysis

	Symptom	Separate Vaccine Group n (%)	Combination Vaccine Group n (%)	P value	Staggered Vaccine Group n (%)	P value
		n = 188	n = 198		n = 187
	Rectal temperature
	≥100.4°F	101(53.7)	134(68.0)	.005	87(46.5)	.000
	>101.3°F	30(16.0)	45(22.8)	.095	21(11.2)	.003
	>103.1°F	1(0.5)	5(2.5)	.216	1(0.5)	.216
	Irritability
	Any	157(83.5)	185(93.4)	.002	158(84.5)	.005
	Grade 3⁎	14(7.4)	25(12.6)	.128	14(7.5)	.128
	Drowsiness
	Any	145(77.1)	159(80.3)	.458	131(70.1)	.024
	Grade 3†	8(4.3)	9(4.5)	1.000	6(3.2)	.602
	Loss of appetite
	Any	101(53.7)	116(58.6)	.357	87(46.5)	.019
	Grade 3‡	2(1.1)	3(1.5)	1.000	2(1.1)	1.000
	Pain at any site
	Any	118(62.8)	126(63.6)	.916	98(52.4)	.030
	Grade 3§	8(4.3)	18(9.1)	.068	10(5.3)	.174
	Pain at DTaP-HepB-IPV or DTaP site
	Any	98(52.1)	119(60.1)	.124	95(50.8)	.081
	Grade 3§	3(1.6)	15(7.6)	.007	9(4.8)	.297
	Redness at any site
	Any	125(66.5)	135(68.2)	.745	102(54.5)	.007
	>20 mm diameter	14(7.4)	16(8.1)	.851	20(10.7)	.388
	Redness at DTaP-HepB-IPV or DTaP site
	Any	97(51.6)	116(58.6)	.184	96(51.3)	.183
	>20 mm diameter	8(4.3)	13(6.6)	.373	18(9.6)	.349
	Swelling at any site
	Any	84(44.7)	102(51.5)	.187	70(37.4)	.006
	>20 mm diameter	5(2.7)	16(8.1)	.024	9(4.8)	.219
	Swelling at DTaP-HepB-IPV or DTaP site
	Any	61(32.4)	88(44.4)	.016	64(34.2)	.047
	>20 mm diameter	2(1.1)	14(7.1)	.004	9(4.8)	.395

DTaP indicate diphtheria–tetanus–acellular pertussis vaccine; DTaP-HepB-IPV, diphtheria and tetanus toxoids and acellular pertussis adsorbed, hepatitis B (recombinant), and inactivated poliovirus vaccine.

⁎ Grade 3 = crying that could not be comforted; prevented normal activity. † Grade 3 = drowsiness that prevented normal activity. ‡ Grade 3 = not eating at all. § Grade 3 = cried when limb was moved/spontaneously painful.