Abstract
Background. Administering 2 separate vaccines for seasonal and pandemic influenza was necessary in 2009. Therefore, we conducted a randomized trial of monovalent 2009 H1N1 influenza vaccine (2009 H1N1 vaccine) and seasonal trivalent inactivated influenza vaccine (TIV; split virion) given sequentially or concurrently in previously vaccinated children.
Methods. Children randomized to 4 study groups and stratified by age received 1 dose of seasonal TIV and 2 doses of 2009 H1N1 vaccine in 1 of 4 combinations. Injections were given at 21-day intervals and serum samples for hemagglutination inhibition antibody responses were obtained prior to and 21 days after each vaccination. Reactogenicity and adverse events were monitored.
Results. All combinations of vaccines were safe in the 531 children enrolled. Generally, 1 dose of 2009 H1N1 vaccine and 1 dose of TIV, regardless of sequence or concurrency of administration, was immunogenic in children ≥10 years of age; children <10 years of age required 2 doses of 2009 H1N1 vaccine.
Conclusions. Vaccines were generally well tolerated. The immune responses to 2009 H1N1 vaccine were adequate regardless of the sequence of vaccination in all age groups but the sequence affected titers to TIV antigens. Two doses of 2009 H1N1 vaccine were required to achieve a protective immune response in children <10 years of age.
Clinical Trials Registration. NCT00943202.
The pandemic 2009 H1N1 influenza virus, influenza A/California/7/09 (H1N1), was identified as a novel influenza strain in April 2009 [1–4]. Children and young adults had little preexisting antibody to this virus, suggesting that seasonal influenza vaccines would not provide protection against the new strain [5–7]. Concern for cocirculation of the novel influenza virus with nonpandemic seasonal influenza virus strains led to rapid evaluation of monovalent pandemic 2009 H1N1 influenza vaccines (2009 H1N1 vaccines) in adults and children [8–15]. This trial evaluated whether the receipt of 2009 H1N1 vaccine either concurrent with, prior to, or following licensed seasonal trivalent inactivated influenza vaccine (TIV) affected the reactogenicity or antibody responses of either vaccine in children 1–17 years of age.
METHODS
Vaccine
The novel split-virion 2009 H1N1 vaccine (Sanofi Pasteur; 2 lots) contained 15 µg/0.5 mL of influenza A/California/7/09 (H1N1)-like virus, based on the high-performance liquid chromatography method to test potency; subsequent testing with single radio immunodiffusion found the potency to be 22–25 µg/0.5 mL. The 2009–2010 seasonal influenza vaccine (Sanofi Pasteur) contained 15 µg each of influenza A/Brisbane/59/2007 (H1N1)-like virus, influenza A/Brisbane/10/2007 (H3N2)-like virus, and influenza B/Brisbane/60/2008-like virus. Subjects <36 months old received intramuscular injections of 0.25 mL (1 lot) of each vaccine in the arm or thigh, and subjects 3–17 years old received 0.5 mL (2 lots) of each vaccine in the deltoid muscle.
Subjects and Study Design
Previously primed subjects (ie, subjects who received 2 or more doses of vaccine in the past) [16], aged 1–17 years, were enrolled in a National Institutes of Health–sponsored, randomized, partially blinded, phase 2 vaccine trial conducted at 7 sites throughout the United States. The subjects' parents or legal guardians provided written informed consent and children provided assent when appropriate. The study was approved by the institutional review board of each of the participating sites. Subjects were equally randomized to 4 groups (Table 1), stratified by age (planned 150 subjects per group with 50 subjects per age stratum: 6–<36 months, 3–9 years, and 10–17 years), to receive 1 dose of seasonal TIV and 2 doses of 2009 H1N1 vaccine in 1 of 4 combinations.
Table 1.
Vaccination Schedule Target Stratified by Age
| Vaccination Schedule |
|||
|---|---|---|---|
| Study Group | Day 0 | Day 21 | Day 42 |
| Group 1, H1N1/H1N1/TIV (n = 150) | H1N1a | H1N1 | TIVb |
| Group 2, H1N1 + TIV/H1N1(n = 150) | H1N1 + TIV | H1N1 | None |
| Group 3, H1N1/H1N1 + TIV (n = 150) | H1N1 | H1N1 + TIV | None |
| Group 4, TIV/H1N1/H1N1 (n = 150) | TIV | H1N1 | H1N1 |
Age strata were 6–<36 months, 3–9 years, and 10–17 years. Subjects in group 1 (H1N1/H1N1/TIV) received inactivated 2009 H1N1 influenza vaccine (H1N1) on days 0 and 21, followed by trivalent influenza vaccine (TIV) on day 42. Subjects in group 4 (TIV/H1N1/H1N1) received TIV on day 0, followed by inactivated 2009 H1N1 vaccine on days 21 and 42. Subjects in group 2 (H1N1 + TIV/H1N1) received 2009 H1N1 vaccine concurrent with TIV on day 0 and H1N1 vaccine on day 21. Subjects in Group 3 (H1N1/H1N1 + TIV) received 2009 H1N1 vaccine on day 0 and 2009 H1N1 vaccine concurrent with TIV on day 21.
a The H1N1 vaccine consisted of 15 µg of influenza A/California/7/09 (H1N1) vaccine for all subjects regardless of age.
b TIV was given as recommended in the package insert: 0.25 mL for subjects 36 months old and 0.5 mL for subjects ≥3 years old.
Safety and Immunogenicity
Safety was measured by assessment of reactogenicity for 8 days, adverse events for 21 days after each vaccination, and serious adverse events and new-onset chronic medical conditions for 8 months after first vaccination.
Local and systemic reactogenicity summary rates were analyzed by dichotomizing the most severe response for each event over the 8-day postvaccination period into binary variables (none vs mild, moderate or, severe) and using exact confidenceintervals. Possible dose response relationships were explored using logistic regression. Unsolicited adverse events were reported using rates and exact 95% confidence intervals. Immunogenicity testing included measurement of hemagglutination antibody inhibition (HAI) titers prior to each vaccination and 21 days after the last vaccination. The primary immunogenicity endpoints were the proportions of subjects, stratified by age, with ≥4-fold HAI titer increase and HAI titer of ≥1:40 against the novel 2009 H1N1 virus 21 days after the first dose of the vaccine.
The proportion of subjects achieving ≥4-fold increase in HAI titer, the proportion of subjects achieving an HAI titer of ≥1:40, and geometric mean titers (GMTs) at 21 days after each vaccination are provided. Analyses of GMTs were based on the formula log2, where “titer” itself was the geometric mean of all replicated values for a sample when such replications were available.
Unless otherwise noted, analyses were primarily conducted using analysis of variance for continuous variables, with results occasionally confirmed, but not reported, using standard nonparametric methods. Binary results, such as reactogenicity, or immune response rates were analyzed using logistic regression and contingency table methods. Most analyses were conducted for a specific milestone, such as 8 days after first novel 2009 H1N1 vaccination. The impact of age and number achieving at least a 1:40 titer was conducted with generalized estimating equation logistic regression to simultaneously analyze the results following each vaccination and to account for the correlation induced from the duplicate measures for a single child.
Antibody Assays
Southern Research Institute performed HAI assays for pandemic 2009 H1N1 vaccine. Influenza (H1) reference antiserum (Centers for Disease Control and Prevention [CDC]; lot 07-0102) and 1× Dulbecco phosphate buffer solution were used as the positive and negative controls, respectively. Turkey red blood cells were used in the HAI assay [17]. Cincinnati Children's Hospital Medical Center performed a standard HAI assay using turkey red blood cells. Virus reference strains from the CDC were grown in MDCK cells [18, 19].
Enrollment Criteria
Healthy children aged ≥6 months through 17 years were eligible to participate. All subjects aged ≥6 months through 9 years had to be primed, or received 1 dose of a 2007–2008 or earlier seasonal influenza vaccine. (Subjects who received their first dose of seasonal influenza vaccine for the 2008–2009 influenza season and the second dose of 2009–2010 seasonal influenza vaccine were excluded from participating in this study.) Subjects of childbearing potential had to use adequate contraception for 30 days after the last vaccination.
Subjects were excluded if they had received a licensed 2009–2010 seasonal influenza vaccine or had 1 of the following: egg allergy; immunosuppression; acute illness; axillary temperature of >100°F or oral temperature of ≥101°F within 3 days prior to vaccination; history of Guillain-Barré syndrome; or planned to travel outside of North America within the 63 days after the first vaccination. Subjects were excluded if they had received 1 of the following prior to vaccination: blood products (3 months), an experimental agent (1 month), live vaccines (4 weeks), or inactivated vaccines (2 weeks); or planned to receive vaccines within 21 days following the last study vaccination.
Sample Size
The sample size for this phase 2 study was powered to detect differences in immune response rates without correction for multiple comparisons. It was projected to have 80% power to conduct a 2-tail test with a type I error of 5.0%, between 2 vaccine groups within the same age stratum when the true differences were <27%. The sample was determined to have >95% power to observe 1 or more serious adverse events related to vaccination when the true underlying rate was 0.50%. Within a single age-stratum vaccine schedule-specific group, the power was 78% to observe 1 or more such events when the true rate was 3.0%
RESULTS
Subject Characteristics and Safety Results
Five hundred thirty-one subjects were enrolled from 20 August 2009 through 21 September 2009. Fifty-two percent of the subjects were male, 80% were white, and the median age was 7.3 years (range, 1.0–17.9 years) (Table 2). Seven subjects did not receive the full immunization series due to receipt of vaccine outside the study, severe tenderness at the injection site, parent/guardian refusal [3], confirmed H1N1 influenza virus infection, and exacerbation of preexisting blackouts. There were no serious adverse events attributed to vaccination. Most adverse events were mild to moderate.
Table 2.
Demographic Data by Study Group for All Subgroups Combined
| Characteristics | All Subjects (n = 531) | H1N1/H1N1/TIV (n = 133) | H1N1 + TIV/H1N1 (n = 133) | H1N1/H1N1 + TIV (n = 133) | TIV/H1N1/H1N1 (n = 132) |
|---|---|---|---|---|---|
| Sex | |||||
| Male | 278 (52) | 65 (49) | 62 (47) | 76 (57) | 75 (57) |
| Female | 253 (48) | 68 (51) | 71 (53) | 57 (43) | 57 (43) |
| Ethnicity | |||||
| Non-Hispanic | 486 (92) | 122 (92) | 123 (92) | 120 (90) | 121 (92) |
| Hispanic | 45 (8) | 11 (8) | 10 (8) | 13 (10) | 11 (8) |
| Race | |||||
| American Indian/Alaskan Native | 1 (0) | 0 | 1 (1) | 0 | 0 |
| Asian | 10 (2) | 5 (4) | 2 (2) | 1 (1) | 2 (2) |
| Hawaiian/Pacific Islander | 0 | 0 | 0 | 0 | 0 |
| Black/African American | 53 (10) | 13 (10) | 15 (11) | 11 (8) | 14 (11) |
| White | 426 (80) | 101 (76) | 107 (80) | 112 (84) | 106 (80) |
| Multiracial | 40 (8) | 13 (10) | 8 (6) | 9 (7) | 10 (8) |
| Other or unknown | 1 (0) | 1 (1) | 0 | 0 | 0 |
| Age | |||||
| Mean (SD) | 8.1 (5.1) | 8.0 (5.0) | 8.2 (5.3) | 7.9 (5.3) | 8.5 (5.0) |
| Median (range) | 7.3 (1.0–17.9) | 7.4 (1–17.9) | 6.8 (1.2–17) | 6.2 (1.2–17.7) | 8.4 (1.1–17.4) |
Data are no. (%) of subjects, unless otherwise indicated.
Abbreviations: H1N1, 2009 H1N1 influenza vaccine; TIV, trivalent influenza vaccine.
Local Reactogenicity
Among all subjects, 15 (3%) subjects experienced severe local reactions (erythema, induration, or tenderness) after vaccination (ie, 6, 8, and 1 subject after first, second, and third vaccination, respectively). All the subjects with severe reactions were in the 2 older age strata. After the first vaccination, moderate local reactions occurred in 47 (8.9%) subjects; of these, 19 (14.3%) subjects received H1N1 + TIV/H1N1. After the second vaccination, moderate local reactions occurred in 44 (8.4%) subjects; of these 24 (18.2%) subjects received H1N1/H1N1 + TIV. A total of 16 subjects (6%) experienced moderate local reactions following the third vaccination; 11 (8.5%) and 5 (3.8%) subjects following the TIV and H1N1 injections, respectively (Table 3).
Table 3.
Local and Systemic Reactogenicity Based on Most Severe Response Reported During Follow-up
| Vaccination 1 |
Vaccination 2 |
Vaccination 3 |
||||
|---|---|---|---|---|---|---|
| Age Stratum, Study Group | No. of Subjects in Group | No. (%) of Subjects With Moderate or Severe Reaction | No. of Subjects in Group | No. (%) of Subjects With Moderate or Severe Reaction | No. of Subjects in Group | No. (%) of Subjects With Moderate or Severe Reaction |
| 1–<3 y | ||||||
| H1N1/H1N1/TIV | 35 | 7 (20) | 35 | 3 (9) | TIV only | … |
| H1N1 + TIV/H1N1 | 33 | 5 (15) | 32 | 6 (19) | 0 | … |
| H1N1/H1N1 + TIV | 35 | 5 (14) | 35 | 3 (9) | 0 | … |
| TIV/H1N1/H1N1 | TIV only | … | 28 | 3 (11) | 28 | 4 (14) |
| 36 mo–9 y | ||||||
| H1N1/H1N1/TIV | 49 | 12 (24) | 47 | 9 (19) | TIV only | … |
| H1N1 + TIV/H1N1 | 51 | 7 (14) | 51 | 7 (14) | 0 | … |
| H1N1/H1N1 + TIV | 49 | 12 (24) | 48 | 10 (21) | 0 | … |
| TIV/H1N1/H1N1 | TIV only | … | 51 | 4 (8) | 51 | 3 (6) |
| 10–17 y | ||||||
| H1N1/H1N1/TIV | 49 | 7 (14) | 49 | 11 (22) | TIV only | … |
| H1N1 + TIV/H1N1 | 49 | 12 (24) | 48 | 8 (17) | 0 | … |
| H1N1/H1N1 + TIV | 49 | 7 (14) | 49 | 9 (18) | 0 | … |
| TIV/H1N1/H1N1 | TIV only | … | 52 | 2 (4) | 52 | 5 (10) |
Reactogenicity data are restricted to visits at which an H1N1 influenza vaccination was given and to reactions in the arm given that vaccination.
Abbreviations: H1N1, 2009 H1N1 influenza vaccine; TIV, trivalent influenza vaccine.
Tenderness was the most prevalent local reaction, followed by redness, across all groups and vaccinations. The percentages of subjects in each treatment group who experienced any local reaction in the 8 days after vaccination were similar among all treatment groups and ranged from 82.7% to 87.9% (Figure 1A and 1B).
Figure 1.
A, Rates of local symptoms by maximum severity grade as captured on the 8-day memory aid with associated 95% confidence intervals, with rate of severe response in subjects receiving first and second 2009 H1N1 influenza virus vaccinations. The numbers represent the first or second 2009 H1N1 vaccination. B, Rates of local symptoms by maximum severity grade as captured on the 8-day memory aid with associated 95% confidence intervals, with rate of severe response in subjects receiving seasonal trivalent influenza vaccine (TIV). The numbers represent the first or second 2009 H1N1 vaccination. C, Rates of systemic symptoms by maximum severity grade as captured on the 8-day memory aid with associated 95% confidence intervals, with rate of severe response. The numbers represent the first, second, or third vaccination. Abbreviations: H, 2009 H1N1 vaccine; T, TIV. The data are stratified by age and vaccine group.
Systemic Reactions
Among all subjects, 10 (1.9%) experienced severe systemic reactions after vaccination. Overall, there were no significant differences within any of the age strata for the solicited systemic symptoms after any vaccination (Table 3).
In the 1–<3-year age stratum, irritability was the most common symptom. The proportion of subjects experiencing any systemic complaint in this age stratum was 69.7%–80.0% across the 4 treatment groups or 74.8% for all groups combined. Three subjects, all receiving H1N1 + TIV/H1N1, experienced 1 severe reaction each, including irritability and vomiting after the first vaccination and fever (defined as axillary temperature of 104°F) after the second vaccination (Figure 1C).
In the 3–9-year age stratum, headache was the most common reaction. The proportion of subjects experiencing any systemic complaint was 31.4%–56.9% across the groups or 42.6% for all groups combined. A total of 7 subjects in 3 of the 4 treatment groups experienced severe systemic reactions. In the H1N1/H1N1/TIV group, 2 subjects experienced severe elevated temperature, feverishness, and decrease in general activity after the first vaccination and 1 subject experienced otitis media after the second vaccination. One subject in the H1N1 + TIV/H1N1 group experienced severe feverishness after the second vaccination. In the H1N1/H1N1 + TIV group, after the first vaccination, 1 subject experienced polymerase chain reaction–confirmed H1N1 influenza virus infection and 1 subject experienced severe myalgia, nausea, vomiting, and decreased general activity. One subject experienced severe feverishness, headache, and decreased general activity after the second vaccination (Figure 1C).
In the 10–17-year age stratum, no severe systemic reactions were reported. The most common reactions were headache and malaise across all the vaccination groups after the first vaccination. The proportion of subjects experiencing any systemic complaint was 50.9%–65.3% across the 4 treatment groups or 57.0% for all groups combined (Figure 1C).
2009 H1N1 Vaccine Immunogenicity Results
Primary Immunogenicity Endpoints
The proportion of subjects with ≥4-fold HAI titer increases against the 2009 H1N1 virus 21 days following the first dose of 2009 H1N1 vaccine were not different when compared by vaccination schedule (P > .53; logistic regression controlling for strata). However, there was a significant difference in response by age strata: 21%, 42%, and 90% of subjects in age groups, 1–<3, 3–9, and 10–17 years, respectively, achieved a 4-fold response (P < .0001; regression controlling for vaccine group). Similarly the proportion of subjects achieving a serum HAI titer of ≥1:40 against the 2009 H1N1 virus 21 days following the first dose of 2009 H1N1 vaccine was significantly different between age strata: 21%, 43%, and 93% in age groups 1–<3, 3–9, and 10–17 years, respectively (P values for logistic regression were identical to those for 4-fold response) (Table 4).
Table 4.
Primary Immunogenicity Results
| 1–<3-y Age Stratum |
3–9-y Age Stratum |
10–17-y Age Stratum |
||||
|---|---|---|---|---|---|---|
| Outcome Measure, Vaccine, Dose or Antigen | No. of Evaluable Subjects | Value | No. of Evaluable Subjects | Value | No. of Evaluable Subjects | Value |
| GMT (95% CI) | ||||||
| 2009 H1N1 | ||||||
| Dose 1 | 126 | 12 (9–15) | 191 | 28 (21–35) | 194 | 297 (241–366) |
| Dose 2 | 118 | 71 (56–89) | 183 | 93 (75–114) | 192 | 392 (338–456) |
| TIV | 182 | 197 | ||||
| H1 | 118 | 246 (202–299) | 219 (187–256) | 428 (368–498) | ||
| H3 | 118 | 356 (293–432) | 621 (513–751) | 861 (755–983) | ||
| B | 118 | 20 (15–25) | 77 (63–95) | 121 (103–142) | ||
| Proportion of subjects with 4-fold increase (95% CI) | ||||||
| 2009 H1N1 | ||||||
| Dose 1 | 124 | 21 (14–29) | 191 | 42 (35–49) | 194 | 90 (85–94) |
| Dose 2 | 117 | 78 (69–85) | 183 | 80 (73–85) | 192 | 95 (91–97) |
| TIV | 182 | 196 | ||||
| H1 | 118 | 65 (57–74) | 43 (36–50) | 60 (53–67) | ||
| H3 | 118 | 83 (76–90) | 65 (57–74) | 63 (57–70) | ||
| B | 118 | 26 (18–34) | 53 (46–61) | 63 (56–70) | ||
| Proportion of subjects with titers of ≥1:40 (95% CI) | ||||||
| 2009 H1N1a | ||||||
| Dose 1 | 126 | 21 (14–29) | 191 | 43 (36–50) | 194 | 93 (88–96) |
| Dose 2 | 118 | 78 (69–85) | 183 | 81 (74–86) | 192 | 98 (95–99) |
| TIV | 182 | 19 | ||||
| H1 | 118 | 97 (95–100) | 97 (94–99) | 99 (98–100) | ||
| H3 | 118 | 99 (98–100) | 98 (97–100) | 99 (98–100) | ||
| B | 118 | 33 (25–42) | 77 (71–83) | 91 (87–95) | ||
The primary immunogenicity endpoints were the proportions of subjects, stratified by age, with ≥4-fold hemagglutination antibody inhibition (HAI) titer increase and HAI titer of ≥1:40 against the novel 2009 H1N1 virus 21 days after the first dose of the vaccine.
Abbreviations: CI, confidence interval; GMT, geometric mean titer; TIV, trivalent influenza vaccine.
a Two (1.6%) subjects aged 6–<36 months, 10 (5.2%) subjects aged 36 months to 9 years, and 25 (12.8%) subjects aged 10–17 years had titers of ≥1:40– prior to the initial vaccination.
Secondary Immunogenicity Endpoints
The proportion of subjects with ≥4-fold increase in HAI titer against the 2009 H1N1 virus 21 days following the second dose of 2009 H1N1 vaccine was 78%, 80%, and 95% of subjects in age groups 1–<3, 3–9, and 10–17 years, respectively. The proportion of subjects achieving a serum HAI titer of ≥1:40 against the 2009 H1N1 virus 21 days following the second dose of 2009 H1N1 vaccine was 78%, 81%, and 98% of subjects aged 1–<3, 3–9, and 10–17 years, respectively. Significance testing using logistic regression following dose 2 yielded essentially identical results as those for dose 1; age stratum was a significant determinant of response (P < .0001) whereas vaccine group was not (Table 4).
GMT Responses
The study population had low pre-vaccination titers against the 2009 H1N1 strain (Figure 2). GMT responses 21 days after the first dose of vaccine differed by age stratum (P < .0001 controlling for vaccine group): 9.7–15, 25.1–30.2, and 140.5–432.5 in subjects in age groups 1–<3, 3–9, and 10–17 years, respectively. Results for the group initially co-administered H1N1 + TIV on day 0 were consistently lower than those of subjects receiving 2009 H1N1 vaccine alone in all 3 age strata. However, significant differences by vaccine group were only observed in the 10–17-year age stratum (P < .001 overall following either dose). Further examination of the pairwise group differences indicate that the group receiving the initial co-administered dose (H1N1 + TIV/H1N1) had a significantly lower GMT following either dose of 2009 H1N1 vaccine than in either group receiving non-concomitant 2009 H1N1 vaccine at the first vaccination (P values controlled for 6 pairwise comparisons; P = .002–.04 per vaccination/strata). There is a trend of the H1N1 + TIV/H1N1 group having lower titers than the group receiving TIV at the first vaccination for both doses of 2009 H1N1 vaccine; however, this difference is not statistically significant.
Figure 2.
Geometric mean titers 21 days after each vaccination by vaccination group and age stratum for each of the 4 vaccine strains: pandemic influenza A/California/7/09 (H1N1)-like virus (H1) and seasonal trivalent influenza vaccine (TIV), comprising influenza A/Brisbane/59/2007 (H1N1)-like virus, influenza A/Brisbane/10/2007 (H3N2)-like virus, and influenza B/Brisbane/60/2008-like virus. Abbreviation: HAI, hemagglutination antibody inhibition.
Receipt of the Prior Year's Seasonal Vaccination, Site, and Sex (Data Not Shown)
Six, 23, and 91 subjects did not receive the previous year's vaccine in age groups 1–<3, 3–9, and 10–17 years, respectively. The impact of receipt of the prior year's seasonal influenza vaccination on log (HAI) were analyzed within age strata using simple univariate t tests and using analysis of covariance including vaccine group, prevaccination titer, sex, clinical site, and receipt of influenza vaccine in the 2 older strata, in which there were sufficient nonrecipients to perform this analysis. Comparisons by prior receipt in the youngest age stratum 21 days after both 2009 H1N1 vaccinations were not significant. In the 3–9-year age stratum, subjects who had not received the prior year's vaccine had greater mean responses in univariate analyses (P < .0001 following both 2009 H1N1 vaccinations) and in the analysis of covariance (P = .03 after first 2009 H1N1 vaccine and P = .052 after second 2009 H1N1 vaccine). This finding was not consistent across all study sites, and the number of subjects without the prior year's vaccination was limited. Within the oldest age stratum the univariate comparisons show no difference by prior vaccination; however, the subjects who were not previously vaccinated did significantly better after controlling for the other variables in the analysis of covariance after the second 2009 H1N1 vaccine dose (P < .01). The H1N1 + TIV/H1N1 group in this stratum also had significantly lower antibody responses than did the other 3 groups in the analyses of covariance (P < .01 for each comparison following both 2009 H1N1 vaccine doses).
TIV Immunogenicity Results
GMT Responses (Table 4)
The group receiving 2009 H1N1 vaccine on days 0 and 21 followed by TIV on day 42 had the highest titer for all 3 TIV antigens in all 3 age strata (Figure 2). Statistically significant differences within 2 age strata were noted. For the seasonal H1N1 antigen, in the 3–9-year stratum, the GMTs for the H1N1/H1N1/TIV and the H1N1 + TIV/H1N1 recipients were 350.6 and 162.3, respectively (P = .005); and the GMTs for the H1N1/H1N1/TIV and the H1N1/H1N1 + TIV recipients were 350.6 and 192.5, respectively (P = .052). For the H1N1 antigen, in the 10–17-year stratum, the GMTs for H1N1/H1N1/TIV and H1N1/H1N1 + TIV recipients were 622.1 and 349.6, respectively (P = .04). Significant differences were also found after comparing other seasonal influenza antigens in select age groups. For example, in the 3–9-year stratum, the GMTs against H3N2 for the H1N1/H1N1/TIV and the H1N1 + TIV/H1N1 recipients were 991.5 and 459.1 (P = .03), respectively. For the B antigen, in the 10–17-year stratum, the GMTs for the H1N1/H1N1/TIV and the H1N1/H1N1 + TIV recipients were 86.1 and 164.6 (P = .02), respectively.
Analysis of covariance for TIV strain-specific log (HAI) responses modeled against baseline titer, site vaccine group, clinical site, age stratum, and prior receipt of TIV resulted in significant associations for prevaccination titers, vaccination treatment group, and prior year's vaccination status (P < .01 for each parameter) for all 3 viral strains represented in the 2009–2010 seasonal influenza vaccine. In addition, there were age strata differences for the H1N1 (P < .0001) and B (P = .02) strains. There were no significant differences based on sex. The group receiving TIV after 2 doses of novel 2009 H1N1 vaccine had statistically significantly higher titers than all other groups for H1N1 (P < .001 for each comparison) and H3N2 (P < .03 for each comparison) and higher titers than in the H1N1/H1N1 + TIV group for strain B (P < .0001). In every instance, the group that reported not having received the prior year's vaccination had statistically significantly higher titers than did those who reported receiving the prior year's vaccination (P < .0001). For age, the results for strata are not consistent across all 3 TIV strains (Table 4). The 1–<3-year age stratum had higher titers than the 3–9-year age stratum for H1N1 (P = .03). There were no significant differences for H3N2. The youngest age stratum had lower responses than did both older age strata for the B strain (P < .0001).
DISCUSSION
The 2009–2010 influenza pandemic presented a unique opportunity to evaluate the effect of the timing of administration of the 2 recommended influenza vaccinations in children. The data were crucial in developing policy for the co-administration of these vaccines during that same influenza season and may potentially inform development of recommendations for future pandemic seasons with other influenza viruses. Of note, the potency of the 2009 H1N1 monovalent study vaccine was 22–25 µg/0.5 mL, and although the potency was slightly higher than anticipated, it was not expected to affect the safety of the vaccine.
The vaccines were well tolerated. The majority of local and systemic reactions were mild to moderate. Less than 3% of subjects experienced severe local reactions, all of which occurred in children ≥10 years of age; even fewer subjects experienced severe systemic reactions, and these occurred in children <10 years of age. The infrequent adverse events following the administration of 2009 H1N1 vaccine in children is consistent with findings in multiple trials and in surveillance during a mass vaccination campaign including (1) a trial comparing 2 injections of either 15 µg or 30 µg of hemagglutinin (CSL Biotherapies) of unadjuvanted split-virion vaccine in children aged 6 months to <9 years [20]; (2) a trial in people ≥3 years of age using 8 vaccine formulations from 10 manufacturers of split-virion vaccine (7.5, 15, or 30 µg of hemagglutinin) with or without aluminum hydroxide and whole-virion vaccine (5 or 10 µg of hemagglutinin) with aluminum hydroxide [21]; (3) a trial comparing 2 injections of 3 different doses (7.5, 15, or 30 µg of hemagglutinin) of adjuvanted and unadjuvanted split-virion vaccine (Hualan Biological Bacterin Company) in people 3–77 years of age [9]; and (4) during a mass vaccination program in China using PANFLU.1 (Sinovac Biotech) with 15 µg of unadjuvanted, split-virion vaccine in people ≥4 years of age [8].
Overall, the proportion of subjects with ≥4-fold HAI titer increase after the first dose of 2009 H1N1 vaccine did not vary by vaccination schedule. However, when comparing age strata, the response rate increased with age, ranging from 21% in the youngest age group to 90% in the oldest age group. This difference in the percentage of subjects achieving a 4-fold increase and/or antibody titers of ≥1:40 remained after the second dose, although, the overall percentages of children reaching a positive endpoint were high. The finding of higher titers with increasing age was seen in several but not all studies; Nolan et al [20] (CSL Biotherpies unadjuvanted 2009 H1N1 vaccine) and Arguedas et al [10] (Novartis 2009 H1N1 vaccine with or without adjuvant) reported seroconversion rates after 1 vaccination to be >90% and 70% percent, respectively. An explanation for the different antibody titer response is not clear, but it may be due to previous exposure or the increase in the amount of antigen used.
When multivariate analysis was performed using vaccine group, sex, clinical site, and receipt of the prior year's seasonal influenza vaccine, an effect of receipt of the prior year's seasonal influenza vaccine on 2009 H1N1 antibody titers was noted in subjects in the 3–9-year age stratum (P = .03 and .052 after the first and second vaccinations, respectively), with those not receiving the previous year's TIV achieving a better response. A similar phenomenon was observed in the older age group, with the group reporting no influenza vaccination in the prior year exhibiting higher antibody responses; however, this was only observed after the second 2009 H1N1 vaccine dose (P = .01). These findings are consistent with those of other studies [20]; it is possible that the blunted response was due to interference of preexisting antibody, but this does not completely explain the finding. Nolan et al [20] also found a decrease in antibody response in subjects with higher baseline titers and previous TIV exposure. More recent reports show that the use of adjuvants may increase antibody responses or allow for antigen sparing in children 3–9 years old [10].
It may be difficult to generalize the results found here because the population studied was predominantly white, although it does represent several geographic regions. In addition, children <1 year of age were not enrolled due to lack of priming.
To our knowledge, no other reported studies of pandemic 2009 H1N1 vaccine evaluated sequential and concurrent administration of 2009 pandemic and seasonal influenza vaccines. The 2009 H1N1 vaccine given sequentially or concurrently with 2009–2010 TIV was safe and well tolerated in previously TIV-primed children. Children <10 years of age generally required 2 doses of 2009 H1N1 vaccine to achieve an adequate response or protective antibody titers. Significant differences in HAI responses based on receipt of the previous year's TIV were noted when subjects were stratified by age group and vaccine treatment group, but these differences are not expected to be clinically significant.
One purpose of the study was to assist in setting policy for the co-administration of the 2 vaccines in children, which is supported by the production of adequate 2009 H1N1 typical HAI titers against TIV. In addition, the study supports the continuing practice of priming children who have not received previous vaccine with 2 doses to reach protective antibody titers [22–24]. Overall, the results of the study support the current CDC recommendation to administer 2 doses of influenza vaccine in children <9 years of age. Although this practice remains necessary at this time, a 2-dose regimen may hinder rapid immunization during a pandemic.
Notes
Acknowledgments. We thank all the volunteers without whom this study would not have been possible. Special thanks to Linda Lambert, Katherine Muth, and Richard Gorman at Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases. We acknowledge the extraordinary efforts of the entire staff at the Vaccine and Treatment Evaluation Unit (VTEU) sites, with special thanks to Jan Tennant, Irene Graham, Edwin Anderson, and the staff of the Saint Louis University VTEU; Joseph A. Hilinski, Mark J. Mulligan, Andrea L. Shane, Paul W. Spearman, Kathleen A. Stephens, and the staff of Emory University VTEU; Susan H. Wooton, W. Paul Glezen, Celsa Tajonera, Virginia Mancha, Armando Correa, and the staff of the Baylor College of Medicine VTEU; Kwabena Sarpong, Peggy Haardt, Carrie Harrington, Diane Barrett, Karen Waterman, and the staff of the University of Texas Medical Branch Clinical Trials program, in Galveston; Kristen Buschle, Stacie Wethington, Michelle Dickey, Rebecca Brady, and the staff of the Cincinnati Children's Hospital Medical Center; Thomas Scholz, Christine Ziebold, Oscar Gomez, Natalie Van Waning, and the staff of the University of Iowa VTEU; Logan Haller, Barbara Taggart, Lisa Slappey, and Valerie Johnson at Southern Research Institute; and Bernadette Jolles of EMMES.
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the Clinical and Translational Science Award program or the National Institutes of Health.
Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (contract HHSN272200800003C to S. E. F. and R. B. B., contract NO1-AI-80006 to D. I. B. and M. A. G., VTEU contract HHSN272200800005C to H. L. K. and A. C. R., contract NO1-30063 to D. L. N., VTEU contract N01AI800002 to F. M. M., contract N01-AI-80002 to C. B. T. and R. E. R., contract HHSN272200800013C to H. H. and M. W., and contract HHSN272200800008C to P. L. W. and G. C.); the National Center for Research Resources, National Institutes of Health (grant UL1RR024979); and the Clinical and Translational Science Award program, National Institutes of Health, National Center for Research Resources (PHS grant UL1 RR025008).
Potential conflicts of interest. S. E. F. has been a consultant to Novartis for influenza vaccines. F. M. M. is a member of the Sanofi Pasteur speaker bureau for influenza vaccines. C. B. T. received funding from the Bill and Melinda Gates Foundation for an influenza vaccine study. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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