Randomized Controlled Trial of Dose Response to Influenza Vaccine in Children Aged 6 to 23 Months Danuta M. Skowronski, Travis S.

Hottes, Mei Chong, Gaston De Serres, David W. Scheifele, Brian J. Ward, Scott A. Halperin, Naveed Z. Janjua, Tracy Chan, Suzana Sabaiduc and Martin Petric Pediatrics; originally published online July 18, 2011; DOI: 10.1542/peds.2010-2777

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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright 2011 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.

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Randomized Controlled Trial of Dose Response to Inuenza Vaccine in Children Aged 6 to 23 Months
WHATS KNOWN ON THIS SUBJECT: Infants and toddlers aged 6 to 23 months experience high rates of inuenza hospitalization, highest in those younger than 1 year. In North America, they are recommended to receive inuenza vaccine annually at a per-injection dose half that recommended for older children and adults. WHAT THIS STUDY ADDS: This randomized controlled trial in infants and toddlers shows that compared with 0.25-mL halfdosing, administration of 2 full 0.5-mL doses of trivalent inactivated inuenza vaccine can increase antibody response without increasing reactogenicity in previously unimmunized infants aged 6 to 11 months.
AUTHORS: Danuta M. Skowronski, MD, FRCPC,a Travis S. Hottes, MSc,a Mei Chong, MSc,a Gaston De Serres, MD, PhD,b David W. Scheifele, MD,c Brian J. Ward, MDCM,d Scott A. Halperin, MD,e Naveed Z. Janjua, MD, MSc, DrPH,a Tracy Chan, BSc, RMCCM,a Suzana Sabaiduc, BSc,a and Martin Petric, PhD, FCCMa
aBritish Columbia Centre for Disease Control, Vancouver, British Columbia, Canada; bInstitut National de Sant Publique du Qubec, Qubec, Canada; cVaccine Evaluation Centre, Child and Family Research Institute, Vancouver, British Columbia, Canada; dVaccine Research Unit, Montreal General Hospital, McGill University Health Centre, Montral, Qubec, Canada; and eCanadian Center for Vaccinology, Dalhousie University, IWK Health Centre, Halifax, Nova Scotia, Canada

abstract
OBJECTIVES: We assessed whether 2 full versus 2 half-doses of trivalent inactivated inuenza vaccine (TIV) could improve immunogenicity without increasing reactogenicity in infants (aged 6 11 months) and toddlers (aged 1223 months). METHODS: Previously unimmunized infants and toddlers were separately randomly assigned to receive 2 full (0.5-mL) or 2 half (0.25-mL) doses of 2008 2009 split TIV. Sera were collected at enrollment and at 27 to 45 days after the second injection. Parents recorded adverse events after each injection. The primary immunogenicity outcome was superiority (1-sided, 0.025) of the full versus the half-dose based on a 10% increase in rates of seroprotection (hemagglutination inhibition titer of 40). The primary reactogenicity outcome was fever of 38C (axillary) within 3 days of either injection. RESULTS: In per-protocol analyses, 252 participants (full dose: n 124; half-dose: n 128) were included. In toddlers, postimmunization seroprotection rates exceeded 85% for all 3 vaccine components without signicant difference by dose. In infants, the full dose induced higher responses for all 3 vaccine components, meeting the 10% test of superiority for the H3N2 (75.4% vs 47.6%; 27.8% [95% condence interval (CI): 11.2 44.5]; P .02) and B/Yamagata (70.2% vs 41.3%; 28.9% [95% CI: 11.9 45.9]; P .02) components but not H1N1 (71.9% vs 54.0%; 18.0% [95% CI: 1.0 34.9]; P .2). Rates of fever were not increased among full- versus half-dose recipients in either age group (5.6% vs 12.7% combined). CONCLUSIONS: Administration of 2 full TIV doses may improve immunogenicity without increasing reactogenicity in infants. Current TIV dosing recommendations for young children warrant additional evaluation. Pediatrics 2011;128:e276e289

KEY WORDS inuenza, inuenza vaccine, randomized controlled trial, immunologic dose-response relationship, children, infants, child, inuenza vaccines/immunology, inuenza vaccines/adverse effects, inuenza vaccines/administration, dosage ABBREVIATIONS TIVtrivalent inactivated inuenza vaccine HAhemagglutinin WHOWorld Health Organization HIhemagglutination inhibition MNmicroneutralization GMTgeometric mean titer NMLNational Microbiology Laboratory NAneuraminidase Drs Skowronski, De Serres, Scheifele, Ward, and Halperin designed the study and oversaw data collection; Dr Petric, Ms Sabaiduc, and Ms Chan oversaw and performed laboratory testing; Ms Chong, Mr Hottes, and Drs Skowronski and Janjua analyzed the data; and all the authors had access to the data (including statistical reports and tables), take responsibility for the study, contributed to the writing of the article, and reviewed and approved the nal manuscript. Dr Skowronski serves as the guarantor. This trial has been registered at www.clinicaltrials.gov (identier NCT00710866). www.pediatrics.org/cgi/doi/10.1542/peds.2010-2777 doi:10.1542/peds.2010-2777 Accepted for publication Apr 19, 2011 Address correspondence to Danuta M. Skowronski, MD, FRCPC, Epidemiology Services, British Columbia Centre for Disease Control, 655 W 12th Ave, Vancouver, British Columbia, Canada V5Z 4R4. E-mail: danuta.skowronski@bccdc.ca. (Continued on last page)

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Children younger than 2 years experience high rates of inuenzaassociated hospitalization, estimated to range from 100 to 500 per 100 000 children during epidemic seasons; the highest rates are seen among infants less than 1 year of age.13 On the basis of these rates, advisory committees in North America recommend that infants (aged 6 11 months) and toddlers (aged 1223 months) receive an inuenza vaccine annually.4,5 Although recommendations elsewhere (eg, the United Kingdom) permit adult-dose (0.5 mL) trivalent inactivated inuenza vaccine (TIV) administration in young children,6 advisory committees in North America recommend that one-half the adult dose be administered per injection to children younger than 3 years of age.7,8 The rationale for this recommendation primarily has been the concern that a full dose may be associated with higher rates of fever (up to 50%) and febrile convulsions in young children.9 However, this perception was on the basis of historical observations with whole virus inuenza vaccines.9 Current splitvirus TIVs are much better tolerated, with mild fever reported in 12% or fewer of vaccinated children aged 1 to 5 years. However, only limited data exist for children aged 6 to 23 months.8,1014 In a 2004 Canadian telephone survey of 690 immunized infants and toddlers, the only symptoms reported in more than 5% of the children were fussiness (21%), fever (11%), decreased eating or drinking (8%), and drowsiness (7%).14 Serious adverse events are rare among immunized children aged 6 to 23 months, with febrile convulsion predominating.13 Studies show variable antibody response to the recommended half-dose of TIV in infants and toddlers, particularly the B component.1521 Antibody responses are lowest in infants.20 Vaccine effectiveness estimates for inPEDIATRICS Volume 128, Number 2, August 2011

fants and toddlers are limited and variable.17,2228 Despite an improved safety prole and suboptimal immunogenicity and effectiveness, the recommendation to halve the per-injection dose of TIV for infants and toddlers has persisted for decades without reevaluation. Other studies have evaluated dose response to the inuenza vaccine in older children (aged 7 years) and the elderly, showing improved immunogenicity without greater reactogenicity when antigen content is increased.2932 No studies to date have assessed the impact of increasing to a full 0.5-mL dose of split TIV in infants and toddlers, as is currently the recommended practice for children aged 3 to 9 years.7,8 Here, we report a study to determine whether giving 2 full doses of split TIV to previously unimmunized infants and toddlers can improve immunogenicity without increasing reactogenicity compared with 2 half-doses.

dom assignment was conducted via the Internet. Participants were block randomly assigned by site and age (aged 6 11 months [infants] and 1223 months [toddlers]). Participants received the same (half or full) dose at both visits by intramuscular injection. At enrollment, 3 mL of blood were collected before giving the rst vaccination. The protocol specied a second vaccination 4 to 6 weeks later; at the analysis phase, participants receiving their second vaccination at an interval of 25 to 49 days were included. Collection of 3 mL of blood was scheduled at 30 to 42 days after the second vaccination; blood collected from 27 to 45 days after the second dose were included in the analysis. Antipyretic or analgesic medication use 24 hours before to 7 days after each vaccination was recorded. Assignment blinding extended to investigators and study staff receiving adverse-event information as well as to parents. Study analysts and laboratory personnel remained blinded to dose assignment until the main analysis was complete. Approval was provided by research ethics boards at Health Canada and the afliated institutions of the investigators at each site. Population Participants were aged 6 to 23 months at enrollment. Those considered ineligible were children with a history of laboratory-conrmed inuenza, previous inuenza vaccination, allergy to any vaccine component, bleeding disorder, or compromised immunity, and those who received immune globulin or other blood products or who were scheduled to receive a live vaccine during the study period. Enrollment was delayed for children given a nonlive vaccine within the previous 14 days or live vaccine within the previous 28
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PATIENTS AND METHODS

Study Design A randomized controlled trial called TITRE I (TIV Infant-Toddler Response Evaluation I) was conducted at 5 sites in 3 Canadian provinces (British Columbia, Quebec, and Nova Scotia). Children aged 6 to 23 months were enrolled between September 1 and December 31, 2008, after obtaining written informed consent from the parent or guardian. At enrollment, participants were randomly assigned to receive 2 spaced doses of 0.25 mL (half-dose) or 0.5 mL (full dose) of a 2008 2009 commercially available TIV, provided free by Sano Pasteur (Lyon, France) (Vaxigrip) and containing 15 g hemagglutinin (HA) of each of the following World Health Organization (WHO)recommended antigens per 0.5 mL: A/Brisbane/10/07 (H3N2); A/Brisbane/ 59/07 (H1N1); and B/Florida/4/06 (Yamagata lineage). Centralized ran-

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days and those who had febrile illness within the previous 72 hours. Immunogenicity Antibody titers were measured using representative noninactivated reference virus strains for each of the 3 vaccine components, as well as B/Brisbane/60/08 of the alternate (B/ Victoria) lineage (the subsequent 2009 2010 TIV component) (Appendix 1). Paired sera were tested in duplicate by hemagglutination inhibition (HI) and microneutralization (MN) assays at the British Columbia Centre for Disease Control in Vancouver per standard protocol (Appendix 1). HI and MN titers were summarized at the subject level as the geometric mean titer (GMT) of duplicate tests. Titers less than 10 were assigned a value of 5; titers were truncated at 10 240. Participants missing paired sera for a given strain were excluded from analyses for that strain. Group GMTs, GMT ratios (postimmunization GMT to preimmunization GMT), seroprotection rates (proportion with HI titers 40),33,34 and seroconversion rates (proportion with preimmunization HI titers 10 and more than fourfold postimmunization rise, or with titers less than 10 preimmunization increasing to 40 postimmunization)33 were estimated for each group and explored by MN. Reactogenicity Parents were given a thermometer, a device to measure local reactions, and a memory aid to record solicited and unsolicited adverse events after immunization for days 0 to 7 after each injection. Solicited adverse events included bedtime axillary temperature, irritability, sleep disturbance, drowsiness, decreased appetite and local tenderness, pain, redness, swelling, and induration. Parents were also asked to record the highe278 SKOWRONSKI et al

est temperature taken at any other time of the day; the highest daily temperature was included in the analysis. At 2 to 3 and 8 to 10 days after each dose, study personnel contacted the parent to collect adverseevent information recorded in the memory aid. Symptoms were categorized using 4-point scales (none, mild, moderate, or severe) described on the memory aid and indicated in Appendix 2. Parents were instructed to report any serious adverse events throughout the study period and were queried at each contact. Serious adverse events were further assessed for causality. Statistical Methods Initial sample-size calculations (1sided; 0.05; 0.20) indicated that 150 participants per group would enable detection of increases of at least 10% in seroprotection rates for inuenza A components (from 80% to 90%), 15% for the inuenza B components (from 60% to 75%), and 10% for fever (from 10% to 20%). To be conservative, superiority was assessed on the basis of the 1-sided test, with 0.025 corresponding to a 95% condence interval around differences. The primary immunogenicity outcome was test of superiority of the full- versus half-dose of TIV on the basis of HI assay using the Wald method for 10% increase in sero-protection rate for each component. The primary reactogenicity outcome was a test of a less than 10% increase between the full dose versus the half-dose based on the Wald method for rate of axillary temperature 38C or higher within 3 days after either injection. Per-protocol and intention-to-treat analyses were performed; with participant attrition of less than 10% in both groups, perprotocol analyses are presented. For antibody estimates, 95% condence intervals were calculated using the exact (Clopper-Pearson) method. Compari-

sons were performed using the Fishers exact test for proportions and t test or Mann-Whitney U test for continuous variables.

RESULTS
Study Population Enrollment totaled 267 subjects, but 15 were excluded because of protocol violations (Appendix 3). Baseline characteristics of the 252 participants (n 128 half-dose; n 124 full dose; n 126 infants; n 126 toddlers) included in per-protocol analyses are presented in Table 1. There were slightly more white participants in the halfdose group and more children with underlying conditions (mainly asthma) in the full-dose group but no other significant differences. Immunogenicity HI Assay Preimmunization titers were low for all 3 vaccine strains in both age groups (Table 2 and Fig 1). Overall, postimmunization seroprotection rates for the 3 strains were higher for full-dose compared with half-dose recipients, but the superiority of the full dose could not be concluded in combined infant and toddler analysis (Table 2 and Fig 1). Full-dose minus half-dose differences in seroprotection rates were 10.7% (95% condence interval: 0.121.5) for H1N1, 16.6% (95% condence interval: 5.9 27.2) for H3N2, and 14.0% (95% condence interval: 2.9 25.0) for B/Yamagata. The lower bounds of these 95% condence intervals do not meet the specied 10% superiority margin for any of the vaccine strains. Thus, superiority of the full-dose over the halfdose regimen cannot be concluded in the analysis of infants and toddlers together. Postimmunization responses to the 3 vaccine components were higher in toddlers than in infants for all immu-

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TABLE 1 Baseline Characteristics of Participants Included in the Per-Protocol Analyses

Variable Range of dates, enrollment Range of dates, second immunization Site, n (%) 1 2 3 4 5 Mean age at enrollment, mean (SD), mo Age group, n (%) 611 mo 1223 mo Female, n (%) Ethnicity, n (%) White Asian Other Mean child birth order, mean (SD) Mean total children for primary caregiver, mean (SD) Length, mean (SD), cm Weight, mean (SD), kg Underlying conditions, n (%) Reactive airway disease or asthma, n (%) Temperature at rst dose, mean (SD), C Prophylactic medication 24 h before rst dose, n (%) Prophylactic medication 24 h before second dose, n (%)
a

Full Dose (N 124) Sep 22Dec 29, 2008 Oct 20Feb 3, 2009 30 (24.2) 28 (22.6) 27 (21.8) 30 (24.2) 9 (7.3) 13.2 (5.1) 61 (49.2) 63 (50.8) 63 (50.8) 89 (71.8) 10 (8.1) 25 (20.2) 1.8 (0.8) 1.8 (0.8) 76.8 (6.7) 10.2 (1.7) 9 (7.3) 7 (5.6) 35.9 (0.4) 3 (2.4) 1 (0.8)

Half-Dose (N 128) Sep 22Dec 30, 2008 Oct 20Feb 4, 2009 32 (25.0) 26 (20.3) 28 (21.9) 29 (22.7) 13 (10.2) 12.8 (5.0) 65 (50.8) 63 (49.2) 71 (55.5) 107 (83.6) 10 (7.8) 11 (8.6) 1.8 (0.9) 1.9 (0.9) 75.3 (6.9) 9.9 (1.8) 2 (1.6) 1 (0.8) 35.9 (0.4) 5 (3.9) 3 (2.3)

Overall (N 252) Sep 22Dec 29, 2008 Oct 20Feb 4, 2009 62 (24.6) 54 (21.4) 55 (21.8) 59 (23.4) 22 (8.7) 13.0 (5.0) 126 (50.0) 126 (50.0) 134 (53.2) 196 (77.8) 20 (7.9) 36 (14.3) 1.8 (0.8) 1.8 (0.9) 76.0 (6.8) 10.1 (1.8) 11 (4.4) 8 (3.2) 35.9 (0.4) 8 (3.2) 4 (1.6)

Pa

.938

.317 .708 .528 .028

.778 .672 .165 .318 .030 .040 .599 .722 .622

P value for comparison between the half-dose and full-dose groups (Fishers exact test for categorical variables and Mann-Whitney U test for continuous variables).

nogenicity outcomes (Table 2). However, the full dose was not superior to the half-dose for any vaccine component in the toddler group. Seroprotection rates exceeded 85% for all 3 vaccine components in toddlers regardless of dose received. On the other hand, among infants full doses elicited substantially higher responses for all vaccine components. Seroprotection rates ranged between 40% and 55% in the half-dose group and between 70% and 75% in the fulldose group (Table 2; Fig 1B and C). The full-dose seroprotection rate was statistically superior to the half-dose rate at the 10% margin for the H3N2 ( 27.8% [95% condence interval: 11.2 44.5]; P .02) and B/Yamagata ( 28.9% [95% condence interval: 11.9 45.9]; P .02) antigens but not H1N1 ( 17.96% [95% condence interval: 1.0 34.9]; P .2). Virtually no antibody to the alternate B/Brisbane/60/08 (Victoria lineage) virus was induced in any group (Fig 1). All of the above ndings were
PEDIATRICS Volume 128, Number 2, August 2011

similar by intention-to-treat analyses. Conclusions related to superiority were not altered when the few participants who met seroprotective thresholds for a given component before immunization were excluded. MN Assay Preimmunization MN titers also were low for all strains in both age groups (Table 3; Fig 1). Postimmunization MN responses generally were higher for toddlers than for infants (Table 3; Fig 1B and C). MN seroprotection rates were higher among full-dose compared with half-dose recipients, notably among infants and for the H3N2 component among toddlers. Postimmunization seroprotection and seroconversion rates measured by MN were similar to those measured by HI for the H1N1 and B/Yamagata components, but for H3N2 the responses by MN were appreciably lower than by HI (Tables 2 and 3; Fig 1). The discrepancy between HI and MN seroprotection

rates for the H3N2 component was greater among infants given the full dose (75.4% vs 50.9%; 24.5%) versus the half-dose (47.6% vs 36.5%; 11.1%); the reverse was true for toddlers given the full dose (91.7% vs 82.0%; 9.7%) versus the half-dose (88.1% vs 64.4%; 23.7%). The increase in H3N2 seroprotection rates with full doses was thus greater by HI than MN for infants (HI 27.8% vs MN 14.4%) but smaller by HI than MN for toddlers (HI 3.6% vs MN 17.6%). Vaccine-induced cross-reactivity to the B/Victoria lineage was again virtually nil (Fig 1). Adverse Events After Immunization Primary Reactogenicity Outcome: Fever Among infants, full-dose and half-dose vaccine recipients had axillary temperatures of 38C or higher by day 3 after the rst injection at rates of 1.5% and 1.5%, respectively, and after the sece279

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TABLE 2 Pre- and Postimmunization (27 45 Days After the Second Dose) Titers to 2008 2009 Inuenza Vaccine Viruses (With 95% Condence Intervals
in Parentheses, Where Applicable), as Measured by an HI Assay, Overall, at Age 6 to 11 Months, and at Age 12 to 23 Months
A/Brisbane/59/07 (H1N1)a Full Dose (N 117) (57, 60)b Preimmunization GMT, mo Overall 611 1223 Seroprotection rate, %, mo Overall 611 1223 Postimmunization GMT, mo Overall 611 1223 GMT ratio, mo Overall 611 1223 Seroprotection rate, %, mo Overall 611 1223 Seroconversion rate, %, mo Overall 611 1223
a b

A/Brisbane/10/07 (H3N2)a Full Dose (N 117) (57, 60)b Half-Dose (N 122) (63, 59)b

B/Florida/4/06 (Yamagata)a Full Dose (N 117) (57, 60)b Half-Dose (N 122) (63, 59)b

Half-Dose (N 122) (63, 59)b

6.4 (5.47.6) 5.6 (4.76.7) 7.2 (5.39.8) 5.1 (1.910.8) 1.8 (0.09.4) 8.3 (2.818.4)

5.5 (5.06.2) 5.3 (4.85.8) 5.8 (4.77.1) 2.5 (0.57.0) 1.6 (0.08.5) 3.4 (0.411.7)

6.1 (5.27.1) 5.5 (4.66.5) 6.7 (5.28.6) 3.4 (0.98.5) 1.8 (0.09.4) 5.0 (1.013.9)

5.2 (4.95.5) 5.0 (5.05.0) 5.4 (4.86.1) 0.8 (0.04.5) 0.0 (0.05.7) 1.7 (0.09.1)

6.1 (5.46.9) 5.4 (5.15.8) 6.7 (5.48.5) 4.3 (1.49.7) 0.0 (0.06.3) 8.3 (2.818.4)

6.2 (5.37.1) 5.6 (4.86.6) 6.8 (5.38.7) 4.9 (1.810.4) 1.6 (0.08.5) 8.5 (2.818.7)

78.4 (61.0100.7) 44.1 (32.559.8) 135.3 (95.6191.6) 12.2 (9.815.2) 7.8 (5.810.4) 18.7 (13.925.2) 81.2 (72.987.8) 71.9 (58.583.0) 90.0 (79.596.2) 80.3 (72.087.1) 70.2 (56.681.6) 90.0 (79.596.2)

56.6 (44.671.8) 33.0 (23.945.6) 100.6 (75.1134.7) 10.2 (8.212.7) 6.2 (4.58.5) 17.4 (13.821.9) 70.5 (61.678.4) 54.0 (40.966.6) 88.1 (77.195.1) 70.5 (61.678.4) 54.0 (40.966.6) 88.1 (77.195.1)

79.3 (65.096.8) 55.2 (42.571.7) 111.8 (84.7147.7) 13.0 (10.915.7) 10.0 (7.812.8) 16.8 (12.921.7) 83.8 (75.889.9) 75.4 (62.285.9) 91.7 (81.697.2) 81.2 (72.987.8) 73.7 (60.384.5) 88.3 (77.495.2)

47.3 (38.857.6) 30.5 (23.639.5) 75.4 (58.197.9) 9.1 (7.511.0) 6.1 (4.77.9) 13.9 (10.817.8) 67.2 (58.175.4) 47.6(34.960.6) 88.1 (77.195.1) 67.2 (58.175.4) 47.6 (34.960.6) 88.1 (77.195.1)

82.9 (65.0105.7) 45.7 (35.059.8) 145.9 (102.8206.9) 13.6 (10.817.1) 8.4 (6.211.3) 21.6 (15.929.4) 80.3 (72.087.1) 70.2 (56.681.6) 90.0 (79.596.2) 80.3 (72.087.1) 70.2 (56.681.6) 90.0 (79.596.2)

51.8 (40.865.8) 25.5 (19.134.1) 110.5 (83.6146.1) 8.4 (6.710.6) 4.5 (3.26.3) 16.3 (13.220.1) 66.4 (57.374.7) 41.3 (29.054.4) 93.2 (83.598.1) 65.6 (56.473.9) 39.7 (27.652.8) 93.2 (83.598.1)

Passaged reference viruses (see Appendix 1). Sample size, overall (aged 6 11 months, aged 1223 months).

ond injection at rates of 0% and 9.2%, respectively (Table 4). Among toddlers, corresponding fever rates for full-dose and half-dose recipients were 1.6% and 3.2%, respectively, after the rst injection and 7.9% and 13.1% , respectively, after the second injection (Table 5). Fever rates were slightly higher among toddlers than infants, but these differences were not signicant. Overall, 5.6% of full-dose and 12.7% of halfdose recipients had fevers of 38C or higher by day 3 after either dose, a difference of 7.1% (95% condence interval: 14.2 to 0.04) (Appendix 4). All of the rate differences were signicantly below the allowed 10% increase in reactogenicity for the full dose (P .001 for infant and combined analyses, P .005 for toddlers). Excluding those with the highest temperature measured by an alternate route (all rece280 SKOWRONSKI et al

tally and at second dose) did not alter these ndings (Appendix 4). Findings were similar in the intention-to-treat analyses. Other Adverse Events The most commonly reported local reactions by day 3 after either dose are shown by age group in Tables 4 and 5 and for infants and toddlers combined in Appendix 4. Local reactions generally were less common in infants (Table 4) than toddlers (Table 5) and more common with full doses versus halfdoses, but none of these differences were signicant. In the combined infant and toddler analysis, the most commonly reported local reactions in both dose groups were redness (23% full dose; 21% half-dose) and tenderness (23% full dose; 26% half-dose): few (5%) reported moderate or se-

vere reactions with either dosage. Swelling (15% full dose; 9% half-dose) and induration (14% full dose; 6% halfdose) were less frequently reported but were more common after full doses (respective P values .12 and .06). Irritability, decreased appetite, drowsiness, and sleep disturbance were reported in more than 35% of participants but were infrequently (5% of all participants) rated as severe, and signicant differences between full doses and half-doses were not observed. Most solicited adverse events had onset by day 3 after immunization, with few additional reports to day 7 thereafter (data not presented). Unsolicited adverse events (regardless of causality assessment) are also shown in Tables 4 and 5 and in Appen-

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A
100

HI, half-dose

MN, half-dose

HI, full-dose

MN, full-dose

Percentage of participants with titre 40

90 80 70 60 50 40 30 20 10 0 A/Brisbane/59/07(H1N1)a A/Brisbane/10/07(H3N2)a B/Florida/4/06(Yamagata)a B/Brisbane/60/08(Victoria)a

B
100 90 80 70 60 50 40 30 20 10 0 A/Brisbane/59/07(H1N1)a -10

HI, half-dose

MN, half-dose

HI, full-dose

MN, full-dose

Percentage of participants with titre 40

A/Brisbane/10/07(H3N2)a

B/Florida/4/06(Yamagata)a

B/Brisbane/60/08(Victoria)a

C
100

HI, half-dose

MN, half-dose

HI, full-dose

MN, full-dose

Percentage of participants with titre 40

90 80 70 60 50 40 30 20 10 0 A/Brisbane/59/07(H1N1)a A/Brisbane/10/07(H3N2)a B/Florida/4/06(Yamagata)a B/Brisbane/60/08(Victoria)a

FIGURE 1
Postimmunization (27 45 days after the second dose) seroprotection rates to 2008 2009 inuenza vaccine viruses and B/Brisbane/60/2008, according to study arm, strain, and assay overall (A), 6 to 11months (B), and 12 to 23 months (C). a Passaged reference viruses (see Appendix 1).

PEDIATRICS Volume 128, Number 2, August 2011

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TABLE 3 Pre- and Postimmunization (27 45 Days After the Second Dose) Titers to 2008 2009 Inuenza Vaccine Viruses (With 95% Condence Intervals
in Parentheses, Where Applicable), as Measured by an MN Assay, Overall, at Age 6 to 11 Months, and at Age 12 to 23 Months
A/Brisbane/59/07 (H1N1)a Full Dose (N 117) (56, 61)b Preimmunization GMT, mo Overall 611 1223 Seroprotection rate, %, mo Overall 611 1223 Postimmunization GMT, mo Overall 611 1223 GMT ratio, mo Overall 611 1223 Seroprotection rate, %, mo Overall 611 1223 Seroconversion rate, %, mo Overall 611 1223
a b

A/Brisbane/10/07 (H3N2)a Full Dose (N 118) (57, 61)b Half-Dose (N 122) (63, 59)b

B/Florida/4/06 (Yamagata)a Full Dose (N 118) (57, 61)b Half-Dose (N 122) (63, 59)b

Half-Dose (N 122) (63, 59)b

6.6 (5.48.0) 5.5 (4.66.7) 7.7 (5.510.9) 6.0 (2.411.9) 1.8 (0.09.6) 9.8 (3.720.2)

5.6 (5.06.3) 5.4 (4.95.9) 5.8 (4.77.2) 2.5 (0.57.0) 1.6 (0.08.5) 3.4 (0.411.7)

6.9 (6.08.0) 6.3 (5.47.4) 7.5 (5.99.5) 3.4 (0.98.5) 1.8 (0.09.4) 4.9 (1.013.7)

6.2 (5.86.7) 6.5 (6.07.0) 6.0 (5.26.9) 0.8 (0.04.5) 0.0 (0.05.7) 1.7 (0.09.1)

5.7 (5.16.4) 5.2 (4.95.5) 6.2 (5.17.7) 2.5 (0.57.3) 0.0 (0.06.3) 4.9 (1.013.7)

5.9 (5.26.8) 5.5 (4.76.3) 6.5 (5.28.1) 4.9 (1.810.4) 1.6 (0.08.5) 8.5 (2.818.7)

124.8 (90.2172.5) 57.6 (39.184.9) 253.5 (161.9397.0) 19.0 (14.524.9) 10.4 (7.315.0) 32.9 (23.246.6) 76.9 (68.284.2) 62.5 (48.575.1) 90.2 (79.896.3) 76.1 (67.383.5) 60.7 (46.873.5) 90.2 (79.896.3)

78.9 (59.6104.4) 43.0 (29.562.6) 150.9 (105.6215.6) 14.1 (11.018.2) 8.0 (5.611.4) 25.9 (19.234.9) 69.7 (60.777.7) 54.0 (40.966.6) 86.4 (75.094.0) 69.7 (60.777.7) 54.0 (40.966.6) 86.4 (75.094.0)

84.3 (65.5108.5) 48.9 (35.966.6) 140.4 (98.4200.3) 12.2 (9.615.5) 7.7 (5.810.2) 18.8 (13.126.8) 66.9 (57.775.3) 50.9 (37.364.4) 82.0 (70.090.6) 66.1 (56.874.6) 50.9 (37.364.4) 80.3 (68.289.4)

44.7 (36.355.1) 29.9 (23.338.3) 68.7 (50.493.7) 7.2 (5.88.9) 4.6 (3.56.0) 11.5 (8.515.5) 50.0 (40.859.2) 36.5 (24.749.6) 64.4 (50.976.4) 50.0 (40.859.2) 36.5 (24.749.6) 64.4 (50.976.4)

109.9 (82.8145.8) 54.2 (37.977.5) 212.6 (146.7308.0) 19.3 (14.725.2) 10.5 (7.215.2) 34.1 (24.447.6) 78.0 (69.485.1) 64.9 (51.177.1) 90.2 (79.896.3) 78.0 (69.485.1) 64.9 (51.177.1) 90.2 (79.896.3)

61.3 (46.980.0) 29.9 (21.441.6) 131.8 (94.5183.8) 10.3 (8.013.3) 5.5 (3.87.8) 20.4 (15.626.5) 64.8 (55.673.2) 44.4 (31.957.5) 86.4 (75.094.0) 63.9 (54.772.4) 42.9 (30.556.0) 86.4 (75.094.0)

Passaged reference viruses (see Appendix 1). Sample size, overall (age 6 11 months, age 1223 months).

dix 4 for infants and toddlers combined. A signicant difference in the rates of unsolicited adverse events was observed in combined analysis only for the miscellaneous category (higher for half-dose), none of which would explain the between-group fever differential. One serious adverse event was reported: a toddler in the halfdose group was hospitalized with pneumonia 28 days after the rst vaccination. The event was deemed unlikely related to the vaccine. Parent Acceptability At the time of nal blood draw, parents (still blinded) were asked to rate the acceptability of the dose and schedule their child received by indicating agreement on a 4-point Likert scale with the following statement: Given my childs experience with this vace282 SKOWRONSKI et al

cine, and if I had another child of the same age, I would be comfortable with him/her receiving the same vaccine and doses. Fifty-four percent of parents whose child received the half-dose regimen strongly agreed, and 46% agreed with this statement. Likewise, 53% of parents whose child received the full dose strongly agreed with this statement, and 46% agreed (P .95). Only 1 parent, whose child received the full dose, disagreed.

were more than 10% higher for all TIV components among children aged 6 to 11 months and were statistically superior for 2 of 3 vaccine components (H3N2: 28% and B/Yamagata: 29%). We did not observe a similar increase in HI antibody responses in toddlers, among whom seroprotection rates exceeded 85% in both dosing groups. Meanwhile, rates of fever were not increased among full-dose recipients, infants, or toddlers, and parent acceptability of increased dosing was likewise comparable. As noted by at least 1 other study,20 vaccine-induced antibody response was substantially lower among infants younger than 1 year of age, the pediatric group at highest risk of inuenzarelated hospitalization and death.2,35 Possible interference from persistent

DISCUSSION
In this randomized controlled trial, we found increased antibody responses among previously unimmunized infants given 2 full doses rather than half-doses of the 2008 2009 TIV. Seroprotection rates in the full-dose group

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TABLE 4 Adverse Events by Day 3 After Immunization: Infants Aged 6 to 11 Months

Dose 1 Full Dose, n 61 Adverse event, n (%) Any event Solicited Unsolicitedb Solicited adverse events after immunization Redness Moderate to severec Severed Swelling Moderate to severec Severed Induration Moderate to severec Severed Tenderness Moderate to severee Severee Fever 37.5C 38C 39C Excluding rectal/tympanic Fever 37.5C 38C 39C Irritability Moderate to severee Severee Decreased appetite Moderate to severee Severee Drowsiness Moderate to severee Severee Sleep disturbance Moderate to severee Severee Unsolicited adverse events after immunizationb Upper respiratory Ocular Gastrointestinal Rash Teething Miscellaneous 44 (72.1) 43 (70.5) 12 (19.7) 10 (16.4) 0 (0.0) 0 (0.0) 5 (8.2) 0 (0.0) 0 (0.0) 3 (4.9) 0 (0.0) 0 (0.0) 10 (16.4) 1 (1.6) 0 (0.0) 1 (1.6) 1 (1.6) 0 (0.0) Half-Dose, n 65 51 (78.5) 48 (73.8) 17 (26.2) 7 (10.8) 0 (0.0) 0 (0.0) 2 (3.1) 0 (0.0) 0 (0.0) 3 (4.6) 0 (0.0) 0 (0.0) 9 (13.8) 0 (0.0) 0 (0.0) 1 (1.5) 1 (1.5) 0 (0.0) Pa Full Dose, n 61 38 (62.3) 36 (59.0) 6 (9.8) 12 (19.7) 0 (0.0) 0 (0.0) 5 (8.2) 0 (0.0) 0 (0.0) 7 (11.5) 0 (0.0) 0 (0.0) 7 (11.5) 0 (0.0) 0 (0.0) 1 (1.6) 0 (0.0) 0 (0.0) Dose 2 Half-Dose, n 65 36 (55.4) 34 (52.3) 15 (23.1) 3 (4.6) 0 (0.0) 0 (0.0) 1 (1.5) 0 (0.0) 0 (0.0) 1 (1.5) 0 (0.0) 0 (0.0) 8 (12.3) 2 (3.1) 0 (0.0) 7 (10.8) 6 (9.2) 3 (4.6) Pa Full Dose, n 61 50 (82.0) 49 (80.3) 17 (27.9) 15 (24.6) 0 (0.0) 0 (0.0) 7 (11.5) 0 (0.0) 0 (0.0) 8 (13.1) 0 (0.0) 0 (0.0) 13 (21.3) 1 (1.6) 0 (0.0) 2 (3.3) 1 (1.6) 0 (0.0) Either Dose Half-Dose, n 65 54 (83.1) 51 (78.5) 28 (43.1) 9 (13.8) 0 (0.0) 0 (0.0) 3 (4.6) 0 (0.0) 0 (0.0) 3 (4.6) 0 (0.0) 0 (0.0) 13 (20.0) 2 (3.1) 0 (0.0) 7 (10.8) 7 (10.8) 3 (4.6) Pa

.535 .695 .406 .438 NA NA .262 NA NA 1.000 NA NA .805 .484 NA 1.000 1.000 NA

.472 .478 .057 .012 NA NA .107 NA NA .029 NA NA 1.000 .497 NA .063 .028 .245

1.000 .829 .095 .173 NA NA .196 NA NA .119 NA NA 1.000 1.000 NA .166 .063 .245

1 (1.6) 1 (1.6) 0 (0.0) 33 (54.1) 18 (29.5) 1 (1.6) 15 (24.6) 2 (3.3) 0 (0.0) 24 (39.3) 2 (3.3) 0 (0.0) 26 (42.6) 11 (18.0) 0 (0.0) 4 (6.6) 0 (0.0) 1 (1.6) 1 (1.6) 6 (9.8) 0 (0.0)

1 (1.5) 1 (1.5) 0 (0.0) 35 (53.8) 18 (27.7) 2 (3.1) 15 (23.1) 5 (7.7) 1 (1.5) 21 (32.3) 5 (7.7) 1 (1.5) 25 (38.5) 15 (23.1) 5 (7.7) 6 (9.2) 1 (1.5) 2 (3.1) 1 (1.5) 6 (9.2) 3 (4.6)

1.000 1.000 NA 1.000 .846 1.000 1.000 .441 1.000 .459 .441 1.000 .717 .516 .058 .745 1.000 1.000 1.000 1.000 .245

1 (1.6) 0 (0.0) 0 (0.0) 20 (32.8) 9 (14.8) 0 (0.0) 9 (14.8) 0 (0.0) 0 (0.0) 16 (26.2) 3 (4.9) 0 (0.0) 19 (31.1) 7 (11.5) 0 (0.0) 4 (6.6) 0 (0.0) 0 (0.0) 1 (1.6) 2 (3.3) 0 (0.0)

4 (6.2) 3 (4.6) 1 (1.5) 27 (41.5) 15 (23.1) 1 (1.5) 19 (29.2) 4 (6.2) 0 (0.0) 17 (26.2) 7 (10.8) 1 (1.5) 22 (33.8) 17 (26.2) 2 (3.1) 7 (10.8) 0 (0.0) 3 (4.6) 0 (0.0) 5 (7.7) 1 (1.5)

.366 .245 1.000 .359 .263 1.000 .057 .12 NA 1.000 .326 1.000 .85 .043 .497 .532 NA .245 .484 .441 1.000

2 (3.3) 1 (1.6) 0 (0.0) 36 (59.0) 21 (34.4) 1 (1.6) 21 (34.4) 2 (3.3) 0 (0.0) 28 (45.9) 4 (6.6) 0 (0.0) 34 (55.7) 16 (26.2) 0 (0.0) 8 (13.1) 0 (0.0) 1 (1.6) 2 (3.3) 8 (13.1) 0 (0.0)

4 (6.2) 4 (6.2) 1 (1.5) 41 (63.1) 25 (38.5) 2 (3.1) 29 (44.6) 8 (12.3) 1 (1.5) 28 (43.1) 12 (18.5) 2 (3.1) 31 (47.7) 25 (38.5) 5 (7.7) 12 (18.5) 1 (1.5) 5 (7.7) 1 (1.5) 10 (15.4) 4 (6.2)

.681 .366 1.000 .716 .712 1.000 .277 .097 1.000 .858 .061 .497 .379 .183 .058 .471 1.000 .209 .610 .802 .120

NA indicates not applicable. a Fishers exact test. b Includes all unsolicited adverse events after immunization, regardless of causality assessment. c Moderate to severe reaction refers to maximum measurement of more than 20 mm. d Severe reaction refers to maximum measurement of 50 mm or more. e See Appendix 3 for scale.

maternal antibody is not supported by the low preimmunization titers we observed in these infants. Immaturity of the immune system may instead contribute to suboptimal vaccine responses in infants. This nding also
PEDIATRICS Volume 128, Number 2, August 2011

may relate to a lack of previous exposure to the inuenza virus early in life, although baseline titers measured in this study also were low for children older than 1 year of age. Current dosing recommendations are historically

predicated on boosting preexisting immunity in adults and older children primed through previous natural exposures, a prole less certain in the very young.9 In our study, GMTs and GMT ratios were slightly higher, but ree283

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TABLE 5 Adverse Events by Day 3 After Immunization: Toddlers Aged 12 to 23 Months

Dose 1 Full Dose (N 63) Adverse event, n (%) Any event Solicited Unsolicitedc Solicited adverse events after immunization Redness Moderate to severed Severee Swelling Moderate to severed Severee Induration Moderate to severec Severee Tenderness Moderate to severef Severef Fever 37.5C 38C 39C Excluding rectal/tympanic Fever 37.5C 38C 39C Irritability Moderate to severef Severef Decreased appetite Moderate to severef Severef Drowsiness Moderate to severef Severef Sleep disturbance Moderate to severef Severef Unsolicited adverse events after immunizationc Upper Respiratory Ocular Gastrointestinal Rash Teething Miscellaneous 45 (71.4) 43 (68.3) 9 (14.3) 10 (15.9) 0 (0.0) 0 (0.0) 10 (15.9) 0 (0.0) 0 (0.0) 6 (9.5) 0 (0.0) 0 (0.0) 11 (17.5) 2 (3.2) 0 (0.0) 2 (3.2) 1 (1.6) 0 (0.0) Half-Dose (N 63) 47 (77.0) 42 (68.9) 11 (18.0) 13 (20.6) 0 (0.0) 0 (0.0) 5 (7.9) 1 (1.6) 1 (1.6) 3 (4.8) 0 (0.0) 0 (0.0) 18 (28.6) 1 (1.6) 0 (0.0) 5 (7.9) 2 (3.2) 0 (0.0) Pa Full Dose (N 63) 45 (71.4) 44 (69.8) 11 (17.5) 6 (9.5) 0 (0.0) 0 (0.0) 5 (7.9) 0 (0.0) 0 (0.0) 5 (7.9) 0 (0.0) 0 (0.0) 9 (14.3) 3 (4.8) 0 (0.0) 7 (11.1) 5 (7.9) 2 (3.2) Dose 2 Half-Dose (N 61)b 39 (63.9) 39 (63.9) 8 (13.1) 12 (19.7) 0 (0.0) 0 (0.0) 5 (8.2) 1 (1.6) 0 (0.0) 3 (4.9) 0 (0.0) 0 (0.0) 10 (16.4) 1 (1.6) 0 (0.0) 10 (16.4) 8 (13.1) 2 (3.3) Pa Full Dose (N 63) 58 (92.1) 56 (88.9) 18 (28.6) 13 (20.6) 0 (0.0) 0 (0.0) 12 (19.0) 0 (0.0) 0 (0.0) 9 (14.3) 0 (0.0) 0 (0.0) 15 (23.8) 4 (6.3) 0 (0.0) 9 (14.3) 6 (9.5) 2 (3.2) Either Dose Half-Dose (N 61)b 53 (86.9) 50 (82.0) 17 (27.9) 17 (27.9) 0 (0.0) 0 (0.0) 8 (13.1) 1 (1.6) 1 (1.6) 5 (8.2) 0 (0.0) 0 (0.0) 20 (32.8) 2 (3.3) 0 (0.0) 12 (19.7) 9 (14.8) 2 (3.3) Pa

.541 1.000 .631 .645 NA NA .271 1.000 1.000 .491 NA NA .204 1.000 NA .44 1.000 NA

.443 .568 .620 .131 NA NA 1.000 .492 NA .718 NA NA .806 .619 NA .442 .392 1.000

.392 .316 1.000 .404 NA NA .466 .492 .492 .396 NA NA .320 .680 NA .479 .419 1.000

2 (3.2) 1 (1.6) 0 (0.0) 27 (42.9) 7 (11.1) 1 (1.6) 17 (27.0) 4 (6.3) 2 (3.2) 17 (27.0) 1 (1.6) 0 (0.0) 23 (36.5) 11 (17.5) 0 (0.0) 3 (4.8) 0 (0.0) 3 (4.8) 1 (1.6) 3 (4.8) 0 (0.0)

5 (7.9) 2 (3.2) 0 (0.0) 25 (39.7) 10 (15.9) 0 (0.0) 18 (28.6) 4 (6.3) 0 (0.0) 19 (30.2) 3 (4.8) 0 (0.0) 22 (34.9) 7 (11.1) 0 (0.0) 1 (1.6) 0 (0.0) 4 (6.3) 3 (4.8) 1 (1.6) 2 (3.2)

.440 1.000 NA .857 .603 1.000 1.000 1.000 .496 .844 .619 NA 1.000 .446 NA .619 NA 1.000 .619 .619 .496

6 (9.5) 4 (6.3) 2 (3.2) 28 (44.4) 10 (15.9) 1 (1.6) 18 (28.6) 8 (12.7) 1 (1.6) 10 (15.9) 4 (6.3) 1 (1.6) 24 (38.1) 11 (17.5) 4 (6.3) 5 (7.9) 0 (0.0) 4 (6.3) 1 (1.6) 2 (3.2) 0 (0.0)

9 (14.8) 7 (11.5) 2 (3.3) 22 (36.1) 11 (18.0) 0 (0.0) 19 (31.1) 6 (9.8) 1 (1.6) 11 (18.0) 2 (3.3) 0 (0.0) 26 (42.6) 11 (18.0) 0 (0.0) 2 (3.3) 0 (0.0) 4 (6.6) 0 (0.0) 1 (1.6) 1 (1.6)

.419 .359 1.000 .365 .813 1.000 .845 .778 1.000 .813 .68 1.000 .715 1.000 .119 .440 NA 1.000 1.000 1.000 .492

8 (12.7) 5 (7.9) 2 (3.2) 38 (60.3) 15 (23.8) 2 (3.2) 27 (42.9) 11 (17.5) 2 (3.2) 21 (33.3) 5 (7.9) 1 (1.6) 34 (54.0) 19 (30.2) 4 (6.3) 8 (12.7) 0 (0.0) 7 (11.1) 2 (3.2) 5 (7.9) 0 (0.0)

11 (18.0) 8 (13.1) 2 (3.3) 36 (59.0) 18 (29.5) 0 (0.0) 26 (42.6) 9 (14.8) 1 (1.6) 25 (41.0) 4 (6.6) 0 (0.0) 33 (54.1) 15 (24.6) 0 (0.0) 3 (4.9) 0 (0.0) 7 (11.5) 3 (4.9) 2 (3.3) 3 (4.9)

.462 .392 1.000 1.000 .544 .496 1.000 .808 1.000 .458 1.000 1.000 1.000 .549 .119 .206 NA 1.000 .677 .440 .116

NA indicates not applicable. a Fishers exact test. b Denominators differ because of missing data. c Includes all unsolicited adverse events after immunization, regardless of causality assessment. d Moderate to severe reaction refers to maximum measurement of more than 20 mm. e Severe reaction refers to maximum measurement of 50 mm or more. f See Appendix 3 for scale.

sponses were otherwise comparable between full-dose and half-dose recipients aged 12 to 23 months. The minimal antibody advantage conferred by the full dose in toddlers is consistent
e284 SKOWRONSKI et al

with recent dose-sparing studies36,37 in young adults in whom comparable antibody responses also have been found with half-doses or full doses. On the other hand, superior re-

sponse to the full dose in infants highlights the need for additional studies of improved vaccine dosing and/or formulations for such young children.

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In this study, we found virtually no antibody induced to the alternate B/Victoria lineage that was not included as a 2008 2009 TIV component. This suggests that exposing immunologically nave infants to TIV containing 1 B/lineage is insufcient to induce priming to the other B/lineage, an observation that requires additional evaluation. It has been suggested that ether treatment of the viral antigen used for HI testing can enhance sensitivity for inuenza B responses but at the expense of specicity.38,39 Absent internationally standardized protocols or validated correlation with clinical protection, the use of ether in some laboratories but not others, sometimes specied in the methods and sometimes not, can complicate the interpretation and comparison across published immunogenicity data. For these results, we did not ether treat the viral antigen used for HI testing, but the same approach was used for all relevant comparisons. The use of noninactivated viruses further supported MN comparisons. Postimmunization results were comparable by HI and MN for the H1N1 and B/Yamagata vaccine components but not the H3N2 component. The pattern of the full-dose versus half-dose increase for H3N2 became less marked in infants but more so in toddlers when measured by MN versus HI. A degree of laboratory variability in inuenza antibody assay results is widely recognized. Postimmunization HI and MN responses typically are well-correlated for homologous viruses,40 but vaccineinduced HI responses may exceed MN for heterologous strains.41 Sequencing of the HA protein of the A/Brisbane/ 10/07 (H3N2) strain used after serial passage in our study showed few amino acid differences at antigenic sites (Leu210Pro and Asn112Ser in sites B and D, respectively) compared with the posted WHO reference strain.
PEDIATRICS Volume 128, Number 2, August 2011

The viral antigen used in our assay did not lead to reduced titers when tested with control ferret anti-sera, suggesting that these mutations did not have a major impact on the observed results (Appendix 1). There are several limitations to this study. The target sample size was not achieved at 1 site, limiting power and interpretation. However, 2 striking trends were apparent, including both dose-response and age-related differences that were consistent across all 3 vaccine components by both HI and MN assays. The consistency of these data argues strongly against their being chance ndings. By convention, we dened seroprotection rates on the basis of a threshold HI titer of 1:40, but we also explored this on the basis of MN. It should be understood, however, that the 1:40 HI threshold used to approve inuenza vaccines annually has not been specically evaluated for infants and toddlers nor is a seroprotective threshold known for MN. Furthermore, the 1:40 HI threshold corresponds generally to a 50% protective level in adults.33,34 Infants and toddlers who had previously been immunized were excluded, and baseline antibody titers were low; consequently, our results apply to immunologically nave infants and toddlers but may not apply to those who have been previously immunized or infected or to children aged 24 to 35 months for whom half-dosing also is currently recommended. Seasonal inuenza activity was delayed in Canada during the 2008 2009 season, peaking in late February/early March, so that infection is unlikely to have substantially inuenced our ndings.42 The follow-up period in this study was 45 days or less, and caution is required when extrapolating results to longer periods. Finally, fever was dened for this study on the basis of axillary temperature for parental ease and child comfort, but this route generally gives

lower temperature recordings than tympanic or rectal measures. This likely explains the low baseline temperatures we found in both study groups (Table 1). However, the most important comparison was between groups on the basis of the same route of measure. This comparison showed the full-dose regimen did not induce more fever than the half-dose regimen. All other indicators of systemic reaction were consistent with this conclusion, as was parent acceptability.

CONCLUSIONS
Despite a decades-long recommendation to halve the dose of split TIV administered to infants and toddlers, this is the rst study to evaluate dose response on immunogenicity and reactogenicity. By administering 2 0.5-mL doses as given to older children, this study suggests an opportunity to improve protection of highly vulnerable infants, without increase in reactogenicity. Although cost-effectiveness analyses are needed, the incremental cost associated with full dosing would be limited to that of the vaccine itself because such a program change would not require an additional medical visit. Additional studies are needed to conrm these ndings in a larger sample, with additional seasons products, further age stratication, and with longer follow-up to assess antibody duration. Relevance to pandemic as well as seasonal vaccine recommendations should be assessed. In particular, additional investigation of the lower response of infants to the currently recommended half-dose approach is urgently required. If conrmed, these results have signicant implications for inuenza vaccine dosing recommendations in this very young age group.

ACKNOWLEDGMENTS Funding for this study was provided by the Public Health Agency of Cane285

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ada (6273-15-2008/4160872) as well as the Ministre de la Sant et des Services Sociaux du Qubec. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. The views expressed herein do not necessarily represent the REFERENCES
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views of the Public Health Agency of Canada. Vaccine was provided without charge from Sano Pasteur (Vaxigrip). We thank the staff of the study sites for their diligence and Leslie Love for coordination nationally. Yan Li of the National Microbiology Laboratory

kindly provided reference viruses used in the antibody assays. We thank Drs Nicole Le Saux, Monika Naus, and Wendy Vaudry for serving as the data-monitoring safety committee for this trial. We thank Dr Jennifer Gardy for assistance with virus sequencing and interpretation.

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www.ema.e u r o p a . e u / d o c s / e n _ G B / document_library/Scientic_guideline/ 2009/09/WC500003945.pdf. Accessed July 28, 2010 Potter CW, Oxford JS. Determinants of immunity to inuenza infection in man. Br Med Bull. 1979;35(1):69 75 Bhat N, Wright JG, Broder KR, et al. Inuenzaassociated deaths among children in the United States, 20032004. N Engl J Med. 2005;353(24):2559 2567 Engler RJM, Nelson MR, Klote MM et al. Halfvs full-dose trivalent inactivated inuenza vaccine (2004 2005): age, dose and sex effects on immune responses. Arch Intern Med. 2008;168(22):24052414 Treanor J, Keitel W, Belshe R et al. Evaluation of a single dose of half strength inactivated inuenza vaccine in healthy adults. Vaccine. 2002;20(7 8):1099 1105 Monto AS., Maassab F. Ether treatment of type B inuenza virus antigen for the hemagglutination inhibition test. J Clin Microbiol. 1981;13(1):54 57

39. Kendal AP, Cate TR. Increased sensitivity and reduced specicity of hemagglutination inhibition tests with ether-treated inuenza B/Singapore/222/79. J Clin Microbiol. 1983;18(4):930 934 40. de Jong JC, Palache AM, Beyer WEP, Rimmelzwaan GF, Boon ACM, Osterhaus ADME. Haemagglutination-inhibiting antibody to inuenza virus. In: Brown F, Haaheim LR, Schild GC, eds. Laboratory Correlates of Immunity to Inuenza: A Reassessment Developments in Biologicals. Vol 115, Basel, Switzerland: Karger; 2003:6373 41. Ansaldi F, Bacilieri S, Ban F, et al. Neutralizing and hemagglutination-inhibiting activities of antibodies elicited by the 2004 2005 inuenza vaccine against drifted viruses. Clin Vaccine Immunol . 2006;13(1):162164 42. Public Health Agency of Canada. FluWatch weekly reports 2008 09 season. Available at: www.phac-aspc.gc.ca/uwatch/08 09/ index-eng.php. Accessed September 13, 2010

APPENDIX 1: LABORATORY METHODS

Viruses Reference viruses were obtained from the National Microbiology Laboratory (NML) in Winnipeg, Canada, and passaged in Madin Darby canine kidney (MDCK) cells at the British Columbia Centre for Disease Control. A/Brisbane/59/07 (H1N1) was used at passage 2 (P2) and A/Brisbane/ 10/07 (H3N2) was used at P3 for both HI and MN. The HA and neuraminidase (NA) genes of all viruses were sequenced to assess homology with the reference strain. Differences between study viruses and reference strains are summarized below, with residue numbering beginning with the N-terminal methionine residues. The HA protein sequence of the NML A/Brisbane/59/07 (H1N1) virus (P2) differed from the WHO reference strain by 1 amino acid in antigenic region B (Asp/Ile203Asn), and the NA protein sequence was identical to the reference strain. The HA sePEDIATRICS Volume 128, Number 2, August 2011

quence of the NML A/Brisbane/10/07 (H3N2) virus (P3) differed from the WHO reference strain by 2 amino acids in antigenic regions B and D (Leu210Pro and Asn112Ser, respectively), and the NA protein sequence was identical to the reference strain. B/Florida/4/06 (Yamagata) (vaccine component) and B/Brisbane/60/08 (Victoria) (alternate nonvaccine lineage) viruses used for both HI and MN assays were passaged twice (P2) in MDCK cells. The HA sequence of the B/Florida/4/06 (Yamagata) virus (P2) showed a mixture of wild-type genotype (identical to the reference) and mutant genotype at position 429 (Gly429Asp); the NA sequence was identical to the WHO posted reference strain. The HA sequence of the B/Brisbane/60/08 (Victoria) virus (P2) also showed a mix of wild-type genotype (identical to the reference) and mutant genotypes (Thr214Ala and Thr277Ile); the NA sequences were identical.

Deposition of Viral Nucleotide Sequences in GenBank Nucleotide sequences of the above study virus segments were deposited into GenBank under the following accession numbers: NML A/Brisbane/ 59/07 (H1N1): CY065747 (HA), CY065749 (NA); NML A/Brisbane/10/07 (H3N2): CY065751 (HA), CY065752 (NA); NML B/Florida/4/06 (Yamagata): CY073895 (HA), CY073896 (NA); and NML B/Brisbane/60/08 (Victoria): CY073893 (HA), CY073894 (NA). HI Assay All sera were frozen at 20C or less until prepared for testing. In preparation for the HI assay, sera were treated with a receptor-destroying enzyme to remove nonspecic agglutinins, and further hemadsorbed with 50% turkey erythrocytes. Sera were serially diluted beginning at 1:10 with phosphate buffered saline and 25 L of each serum dilution was reacted with 25 L of antigen containing 4 HI units of virus for 30 minutes. To each mixture, 50 L
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of 0.5% turkey erythrocytes were added, and after mixing, the preparations were incubated for 30 minutes. The HI titer was the inverse of the highest dilution at which detectable HI activity still was present. All sera were assayed in duplicate. MN Assay The same study viruses were used for the MN assay as for the HI assay. Viral titers were determined by tissue culture infective dose 50 on the basis of the Karber method.1 The sera were inactivated by heating at 56C for 30 minutes. The sera were serially diluted beginning at 1:10, and to each dilution 100 infectious units of virus were added. The plates were incubated for 2 hours at 37C to allow for virusantibody interaction. The contents of each well were then transferred onto microtiter plates with conuent monolayers of MDCK cells. After 3 hours of additional incubation at 37C, the medium in each well was removed and replaced by fresh medium (MegaVir; HyClone, Logan, Utah) containing L-1-tosylamido-2phenylethyl chloromethyl ketone (TPCK)-treated trypsin. The plate was then again incubated at 37C and monitored for the appearance of cytopathic effects on days 3 and 5. The MN titer was dened as the serum dilution in the well immediately preceding the wells with cytopathic effects. MN was conducted in duplicate up to 2 times until intra-assay concordance was achieved (twofold or less) and GMTs reported. Reference 1. Karber G. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch Exp Path Pharmacol. 1931;162(4):480 483

APPENDIX 2 Participant Flow Chart

APPENDIX 3: RESPONSE SCALES FOR PARENT-MEASURED ADVERSE EVENTS AFTER IMMUNIZATION

Local and systemic reactions were measured for days 0 to 7 after each immunization by parents of study participants. Responses were categorized into 4-point scales (none, mild, moderate, or severe) described as follows:
Axillary temperature: none (37.5C),

when injected limb moved or movement of injected limb reduced).

Irritability: mild (easily consolable),

moderate (requires increased attention), or severe (inconsolable).

Sleep disturbance: mild (less sleep

mild (37.5C to 38.0C), moderate (38.0C to 39.0C), or severe (39.0C).

or woke once more than usual), moderate (less sleep or woke repeatedly), or severe (less sleep or virtually sleepless night).
Drowsiness: mild (noticeable but

Local redness, swelling, or indura-

tion: mild (0 mm but 20 mm), moderate (20 mm and 50 mm), or severe (50 mm).

does not interfere with usual daily activities), moderate (interferes with daily activities), or severe (prevents usual daily activities).
Decreased appetite: mild (eating or

Local pain or tenderness: mild (mi-

nor reaction when injection site touched), moderate (child cries or protests when injection site touched), or severe (child cries

feeding less than usual), moderate (eating or feeding much less than usual), or severe (not eating or feeding at all).

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APPENDIX 4 Adverse Events by Day 3 After Immunization: Infants and Toddlers Combined
Adverse Event Full Dose (N 124), n (%) 89 (71.8) 86 (69.4) 21 (16.9) 20 (16.1) 0 (0.0) 0 (0.0) 15 (12.1) 0 (0.0) 0 (0.0) 9 (7.3) 0 (0.0) 0 (0.0) 21 (16.9) 3 (2.4) 0 (0.0) 3 (2.4) 2 (1.6) 0 (0.0) 3 (2.4) 2 (1.6) 0 (0.0) 60 (48.4) 25 (20.2) 2 (1.6) 32 (25.8) 6 (4.8) 2 (1.6) 41 (33.1) 3 (2.4) 0 (0.0) 49 (39.5) 22 (17.7) 0 (0.0) 7 (5.6) 0 (0.0) 4 (3.2) 2 (1.6) 9 (7.3) 0 (0.0) Dose 1 Half-Dose (N 128), n (%) 98 (77.8) 90 (71.4) 28 (22.2) 20 (15.6) 0 (0.0) 0 (0.0) 7 (5.5) 1 (0.8) 1 (0.8) 6 (4.7) 0 (0.0) 0 (0.0) 27 (21.1) 1 (0.8) 0 (0.0) 6 (4.7) 3 (2.3) 0 (0.0) 6 (4.7) 3 (2.3) 0 (0.0) 60 (46.9) 28 (21.9) 2 (1.6) 33 (25.8) 9 (7.0) 1 (0.8) 40 (31.3) 8 (6.3) 1 (0.8) 47 (36.7) 22 (17.2) 5 (3.9) 7 (5.5) 1 (0.8) 6 (4.7) 4 (3.1) 7 (5.5) 5 (3.9) Pa Full-dose (N 124), n (%) 83 (66.9) 80 (64.5) 17 (13.7) 18 (14.5) 0 (0.0) 0 (0.0) 10 (8.1) 0 (0.0) 0 (0.0) 12 (9.7) 0 (0.0) 0 (0.0) 16 (12.9) 3 (2.4) 0 (0.0) 8 (6.5) 5 (4.0) 2 (1.6) 7 (5.6) 4 (3.2) 2 (1.6) 48 (38.7) 19 (15.3) 1 (0.8) 27 (21.8) 8 (6.5) 1 (0.8) 26 (21.0) 7 (5.6) 1 (0.8) 43 (34.7) 18 (14.5) 4 (3.2) 9 (7.3) 0 (0.0) 4 (3.2) 2 (1.6) 4 (3.2) 0 (0.0) Dose 2 Half-Dose (N 126), n (%)b 75 (59.5) 73 (57.9) 23 (18.3) 15 (11.9) 0 (0.0) 0 (0.0) 6 (4.8) 1 (0.8) 0 (0.0) 4 (3.2) 0 (0.0) 0 (0.0) 18 (14.3) 3 (2.4) 0 (0.0) 17a (13.5) 14b (11.1) 5c (4.0) 13 (10.3) 10 (7.9) 3 (2.4) 49 (38.9) 26 (20.6) 1 (0.8) 38 (30.2) 10 (7.9) 1 (0.8) 28 (22.2) 9 (7.1) 1 (0.8) 48 (38.1) 28 (22.2) 2 (1.6) 9 (7.1) 0 (0.0) 7 (5.6) 0 (0.0) 6 (4.8) 2 (1.6) Pa Full Dose (N 124), n (%) 108 (87.1) 105 (84.7) 35 (28.2) 28 (22.6) 0 (0.0) 0 (0.0) 19 (15.3) 0 (0.0) 0 (0.0) 17 (13.7) 0 (0.0) 0 (0.0) 28 (22.6) 5 (4.0) 0 (0.0) 11 (8.9) 7 (5.6) 2 (1.6) 10 (8.1) 6 (4.8) 2 (1.6) 74 (59.7) 36 (29.0) 3 (2.4) 48 (38.7) 13 (10.5) 2 (1.6) 49 (39.5) 9 (7.3) 1 (0.8) 68 (54.8) 35 (28.2) 4 (3.2) 16 (12.9) 0 (0.0) 8 (6.5) 4 (3.2) 13 (10.5) 0 (0.0) Either Dose Half-Dose (N 126), n (%)b 107 (84.9) 101 (80.2) 45 (35.7) 26 (20.6) 0 (0.0) 0 (0.0) 11 (8.7) 1 (0.8) 1 (0.8) 8 (6.3) 0 (0.0) 0 (0.0) 33 (26.2) 4 (3.2) 0 (0.0) 19 (15.1) 16 (12.5) 5 (4.0) 15 (11.9) 12 (9.5) 3 (2.4) 77 (61.1) 43 (34.1) 2 (1.6) 55 (43.7) 17 (13.5) 2 (1.6) 53 (42.1) 16 (12.7) 2 (1.6) 64 (50.8) 40 (31.7) 5 (4.0) 15 (11.9) 1 (0.8) 12 (9.5) 4 (3.2) 12 (9.5) 7 (5.6) Pa

Any event Solicited Unsolicitedc Solicited AEFIs Redness Moderate-severed Severee Swelling Moderate-severed Severee Induration Moderate-severed Severee Tenderness Moderate-severef Severef Fever 37.5C 38C 39C Excluding rectal/tympanic Fever 37.5C 38C 39C Irritability Moderate-severef Severef Decreased appetite Moderate-severef Severef Drowsiness Moderate-severef Severef Sleep disturbance Moderate-severef Severef Unsolicited AEFIsc Upper respiratory Ocular Gastrointestinal Rash Teething Miscellaneous

.309 .782 .340 1.000 NA NA .075 1.000 1.000 .434 NA NA .426 .364 NA .5 1.000 NA .500 1.000 .900 .759 1.000 1.000 .597 .618 .788 .217 1.000 .698 1.000 .060 1.000 1.000 .749 .684 .613 .060

.240 .302 .389 .579 NA NA .313 1.000 NA .041 NA NA .854 1.000 NA .090 .054 .447 .244 .167 1.000 1.000 .324 1.000 .150 .808 1.000 .878 .797 1.000 .601 .142 .445 1.000 NA .540 .245 .749 .498

.716 .407 .224 .760 NA NA .122 1.000 1.000 .060 NA NA .557 .748 NA .173 .078 .447 .400 .221 1.000 .897 .416 .682 .443 .560 1.000 .701 .206 1.000 .529 .582 1.000 .850 1.000 .486 1.000 .836 .014

AEFI indicates adverse event following immunization; NA, not applicable. a Fishers exact test. b Denominators differ because of missing data. c Includes all unsolicited AEFIs, regardless of causality assessment. d Moderate-severe reaction refers to maximum measurement of more than 20 mm. e Severe reaction refers to maximum measurement of 50 mm. f See Appendix 3 for scale.

(Continued from rst page) PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright 2011 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: Dr De Serres has received research grant funding for unrelated studies from Sano Pasteur and GlaxoSmithKline; Dr Scheifele has received research grant funding for unrelated studies from Sano Pasteur, GlaxoSmithKline, and Wyeth; Dr Ward currently shares a CIHR team grant with GlaxoSmithKline investigators; and Dr Halperin has received research grant funding for unrelated studies from Sano Pasteur, GlaxoSmithKline and Novartis. The other authors have indicated they have no nancial relationships relevant to this article to disclose.

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Randomized Controlled Trial of Dose Response to Influenza Vaccine in Children Aged 6 to 23 Months Danuta M. Skowronski, Travis S. Hottes, Mei Chong, Gaston De Serres, David W. Scheifele, Brian J. Ward, Scott A. Halperin, Naveed Z. Janjua, Tracy Chan, Suzana Sabaiduc and Martin Petric Pediatrics; originally published online July 18, 2011; DOI: 10.1542/peds.2010-2777
Updated Information & Services including high resolution figures, can be found at: http://pediatrics.aappublications.org/content/early/2011/07/13 /peds.2010-2777 This article has been cited by 3 HighWire-hosted articles: http://pediatrics.aappublications.org/content/early/2011/07/13 /peds.2010-2777#related-urls Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://pediatrics.aappublications.org/site/misc/Permissions.xht ml Information about ordering reprints can be found online: http://pediatrics.aappublications.org/site/misc/reprints.xhtml

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