Human Vaccines & Immunotherapeutics

ISSN: 2164-5515 (Print) 2164-554X (Online) Journal homepage: https://www.tandfonline.com/loi/khvi20

Safety and immunogenicity of 2010–2011 A/

H1N1pdm09-containing trivalent inactivated
influenza vaccine in adults previously given AS03-
adjuvanted H1N1 2009 pandemic vaccine
Results of a randomized trial

David W. Scheifele, Marc Dionne, Brian J. Ward, Curtis Cooper, Otto G.

Vanderkooi, Yan Li, Scott A. Halperin & PHAC/CIHR Influenza Research
Network

To cite this article: David W. Scheifele, Marc Dionne, Brian J. Ward, Curtis Cooper, Otto G.
Vanderkooi, Yan Li, Scott A. Halperin & PHAC/CIHR Influenza Research Network (2013) Safety
and immunogenicity of 2010–2011 A/H1N1pdm09-containing trivalent inactivated influenza vaccine
in adults previously given AS03-adjuvanted H1N1 2009 pandemic vaccine, Human Vaccines &
Immunotherapeutics, 9:1, 136-143, DOI: 10.4161/hv.22619

To link to this article: https://doi.org/10.4161/hv.22619

Copyright © 2013 Landes Bioscience Published online: 01 Jan 2013.

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 Research paper

Human Vaccines & Immunotherapeutics 9:1, 136–143; January 2013; © 2013 Landes Bioscience

Safety and immunogenicity of 2010–2011

A/H1N1pdm09-containing trivalent inactivated
influenza vaccine in adults previously given
AS03-adjuvanted H1N1 2009 pandemic vaccine
Results of a randomized trial
David W. Scheifele,1,* Marc Dionne,2 Brian J. Ward,3 Curtis Cooper,4 Otto G. Vanderkooi,5 Yan Li,6 Scott A. Halperin7
and PHAC/CIHR Influenza Research Network8
1
Vaccine Evaluation Center; BC Children’s Hospital; University of British Columbia; Vancouver, BC Canada; 2Unité de Recherche en Santé Publique; Institut nationale de santé
publique du Québec; Québec City, QC Canada; 3Vaccine Study Center; Research Institute of the McGill University Health Centre; Montreal, QC Canada; 4University of Ottawa;
The Ottawa Hospital Division of Infectious Diseases; Ottawa, ON Canada; 5University of Calgary; Alberta Health Services and Alberta Children’s Hospital; Calgary, AB Canada;
6
National Microbiology Laboratory; Winnipeg, MB Canada; 7Canadian Center for Vaccinology; Dalhousie University; Halifax, NS Canada; 8Public Health Agency of Canada/
Canadian Institutes of Health Research Influenza Research Network (PCIRN); Dalhousie University; Halifax, NS Canada

Keywords: immunization, adults, influenza, pandemic, adjuvant, vaccine safety

Abbreviations: BMI, body mass index; CHMP, Committee for Medicinal Products for Human Use; CI, confidence interval;
DSMB, Data safety monitoring board; EMEA, European Medicines Evaluation Agency; GMT, geometric mean titer; HAI,
hemagglutination inhibition; H1N1pdm09, swine-derived A/H1N1 virus responsible for the 2009 pandemic;
ORS, oculorespiratory syndrome; PCIRN, Public Health Agency of Canada/Canadian Institutes of Health Influenza Research
Network; SD, standard deviation; TIV, trivalent inactivated influenza vaccine; VAAE, vaccine-attributable adverse event;
WHO, World Health Organization

Many Canadians received a novel AS03-adjuvanted vaccine during the 2009 influenza A/H1N1 pandemic. Longer term
implications of adjuvant use were unclear: would anti-H1N1 immune responses persist at high levels and, if so, could that
result in increased or unusual adverse effects upon re-exposure to H1N1pdm09 antigen in the trivalent influenza vaccine
(TIV) for 2010–11? To answer these questions, adults given AS03-adjuvanted H1N1pdm09 vaccine (Arepanrix®, GSK Canada)
9–10 mo earlier were enrolled in an evaluator-blinded, crossover trial to receive 2010–2011 non-adjuvanted TIV (Fluviral®,
GSK Canada) and placebo 10 d apart, in random order. Adverse effects were monitored for 7 d after each injection.
Vaccine-attributable adverse event (VAAE) rates were calculated by subtracting rates after placebo from those after
vaccine. Blood was obtained pre-vaccination and 21–30 d afterward to measure hemagglutination inhibiting antibody
titers. In total, 326 participants were enrolled and 321 completed the study. VAAE rates were low except for myalgia
(18.6%) and injection site pain (63.2%). At baseline, H1N1pdm09 titers ≥ 40 were present in 176/325 subjects (54.2%, 95%
confidence interval 48.6, 59.7), with a geometric mean titer (GMT) of 37.4 (95% CI 32.8, 42.6). Post-immunization, 96.0%
(95% CI 92.3, 97.8) had H1N1pdm09 titers ≥ 40, with GMT of 167.4 (95% CI 148.7, 188.5). Responses to both influenza A
strains in TIV were similar, implying no lasting effect of adjuvant exposure. In summary, titers ≥ 40 persisted in only half
the participants 9–10 mo after adjuvanted pandemic vaccine but were restored in nearly all after TIV vaccination, with
minimal increase in adverse effects.

Introduction vaccine, which accounted for over 96% of pandemic vaccine

doses administered. The adjuvant is an oil-in-water emulsion
During the A/H1N1 2009 global pandemic,1 adjuvanted containing squalene and tocopherol. This vaccine was used in
influenza vaccines were widely used for the first time. Along a dose-sparing formulation (3.75 μg hemagglutinin per adult
with many other countries, Canada deployed a novel AS03- dose), one dose of which was adequately immunogenic in naïve
adjuvanted H1N1pdm09 vaccine (Arepanrix®, GSK Canada).2-5 adults.2-5 By the end of the mass campaign in Canada, over 40%
All Canadians older than 6 mo were eligible to receive this of the population had received this vaccine.6 It was not known

*Correspondence to: David W. Scheifele; Email: dscheifele@cfri.ca

Submitted: 09/12/12; Revised: 10/16/12; Accepted: 10/22/12
http://dx.doi.org/10.4161/hv.22619

136 Human Vaccines & Immunotherapeutics Volume 9 Issue 1

 Research paper research paper

Table 1. Characteristics of study participants in a randomized, crossover and placebo one week apart, in random, undisclosed order.
trial of 2010–11 inactivated, trivalent influenza vaccine vs. placebo
Adverse events were documented daily; rates following placebo
First injection: First injection: were subtracted from rates after vaccine to determine vaccine-
Total group
TIV* Placebo*
attributable rates.14,15 The results were used to inform subsequent
Male 48 (29.4%) 48 (29.4%) 96 (29.4%) seasonal vaccination programs in Canada. Secondary objectives
Age, years, mean 41.7 (11.4) 41.5 (11.2) 41.6 (11.3) were to measure residual antibody titers to H1N1pdm09 virus in
(± SD) previously vaccinated adults and the magnitude of the booster
BMI, mean (± SD) 26.9 (5.3) 26.8 (5.5) 26.8 (5.4) response to re-vaccination with this antigen, in comparison to A/
H3N2 responses.
Caucasian/white 145 (89.0%) 145 (89.0%) 290 (89.0%)
Chronic health 89 (54.6%) 87 (53.4%) 176 (54.0%)
Results
condition**

Previous TIV 149 (91.4%) 144 (88.3%) 293 (89.9%) Enrollment totaled 326 subjects, who were predominantly female
Total number 163 163 326 and Caucasian/white (Table 1). Randomized groups were well
*Opposite assignment applied to second (crossover) injections; **Refers matched (Table 1). The rate of prior seasonal influenza vaccina-
to any ongoing medical condition, not just those posing increased risk tion was high as most subjects were employed by or affiliated with
with influenza. the participating health care institutions. Reported chronic health
conditions were generally of minor nature. Centers commenced
at the time if adjuvant use would be associated with persistently enrollment on the same day (9 August 2010) and completed
elevated antibody responses or the rapid decay typically seen after it within 5 d. Compliance with the protocol was high (Fig. 1)
seasonal inactivated vaccines.7 In studies launched while the pan- enabling per protocol analysis of safety and immunogenicity data.
demic vaccine was initially deployed in 2009, evidence of prior Safety observations. As Table 2 shows, TIV vaccination was
infection was already present in one-third of adults under 60 y of associated with higher than background (post-placebo) rates
age.4,5 These primed individuals developed strong booster anti- for myalgia, tiredness, headache, malaise, arthralgia, sleep dis-
body responses to H1N1pdm09 vaccination, with greater poten- turbance and diarrhea during the week after vaccination. Only
tial for persistence. Both natural infection and vaccination with myalgia, tiredness and headache occurred at vaccine-attributable
adjuvant were expected to elicit greater cell-mediated immune rates (VAR) > 5.0%, with myalgia leading at 18.6%. Most sub-
responses than observed following standard trivalent seasonal jects who reported these symptoms rated them as mild/moderate
vaccines. (Table 2). The peak VAR’s for myalgia and headache were reached
In 2010 the A/H1N1pdm09 virus continued to circulate with later during the day of vaccination, at 16.9% and 4.9%, respec-
minimal antigenic change. The World Health Organization rec- tively, while reports of tiredness peaked next day, with a VAR of
ommended that the same H1N1pdm09 vaccine strain be included 5.8%. Reported rates of myalgia and headache then declined to
in the trivalent inactivated vaccine (TIV) for the 2010–2011 match those following placebo from day 3 onwards, with tired-
influenza season.8 Some candidates for re-vaccination with the ness rates normalizing one day later. A similar pattern was seen
H1N1pdm09 strain would have been strongly primed by prior with reports of malaise, arthralgia, sleep disturbance and diar-
infection, vaccination with adjuvant or both. It was certainly rhea. Background rates during the week after placebo were > 5%
conceivable that such individuals might have greater residual cel- for headache, tiredness, myalgia and malaise (Table 2), illustrat-
lular immunity and/or specific antibody levels than ordinarily ing the value of controlled observations for this age group.
encountered after seasonal influenza vaccination and that this At the injection site, pain was reported by 63.2% of par-
might predispose them to increased vaccine reactions. Vaccination ticipants (204/323) during the week after TIV vaccine and by
in the presence of specific cellular immunity has been linked to 8.0% (26/323) after placebo. TIV-related pain was rated as mild
increased injection site reactions after childhood booster doses (186 subjects, 57.6%) or moderate (18 subjects, 5.6%) with no
of adjuvanted, pertussis-containing vaccines9,10 while vaccination instances of severe pain. Local redness followed TIV in 45 par-
in the presence of high titers of antitoxin increased reaction rates ticipants (13.9%), with 38 (11.8%) having mild (< 25 mm) and
after repeated tetanus toxoid boosters.11 It was not known at the 7 (2.2%) having moderate (26–99 mm) redness. Local swelling
time if residual cellular or humoral immunity to H1N1 would followed TIV in 31 participants (9.6%), with a single instance
be sufficient to cause reactions with a first booster dose of H1N1 (0.3%) of severe swelling (≥ 100 mm). Injection site symptoms
antigen. The post-pandemic situation was unique in modern were short-lived: all resolved by Day 6 after vaccination, well
experience with influenza vaccines and warranted investigation, before second injections (data not shown).
especially given the potential for unpredictable variations in TIV A strong association was evident on day 1 after vaccination
reactogenicity such as occurred in children in Western Australia between reported rates of injection site pain and myalgia. Among
in 2010 with a particular TIV formulation.12 145 subjects with pain on that day, 41 (28.3%) also had general-
We conducted a pre-season study to rapidly assess the safety ized myalgia whereas among 180 subjects without pain, only 10
of re-vaccination13 with H1N1pdm09 antigen. Eligible adults (5.6%) reported myalgia (p < 0.0001, Chi- square test).
had received the adjuvanted pandemic vaccine 9–10 mo previ- During the first 24 h after TIV vaccination 32 subjects (9.8%)
ously, from community providers. Participants received TIV reported 47 new-onset respiratory symptoms. These participants

www.landesbioscience.com Human Vaccines & Immunotherapeutics 137

more often reported concurrent myalgia than those
without new respiratory symptoms (34.4% vs. 13.6%,
p < 0.01, odds ratio 3.3, 95% confidence interval 1.5,
7.4). No significant association was detected with
other general symptoms (data not shown). Twenty-
one vaccinated subjects reported a single respira-
tory symptom (sore throat-8, cough-6, hoarseness-3,
wheezing-2, red eyes-2), all of which were rated mild.
Eleven vaccinated subjects (3.4%) had multiple, new-
onset respiratory symptoms (Table 3), more consistent
with oculorespiratory syndrome (ORS).16,17
All symptoms were rated mild or moderate and none
of those affected sought medical attention. During the
first 24 h after placebo injection, 11 subjects (3.4%)
reported 19 new-onset respiratory symptoms, mainly
sore throat (6), hoarseness (3), cough (3), chest tight-
ness (3) and ocular redness (1). The frequency of respi-
ratory symptoms was significantly lower after placebo
than after TIV (3.4% vs. 9.8%, p < 0.001) but the
spectrum of symptoms was similar in both settings.
Only 3 subjects had multiple respiratory symptoms
after placebo (Table 3), yielding a vaccine-attributable
rate of ORS-like symptoms of 2.2% (p < 0.01).
Other notable adverse events included minor
allergy-like reactions (generalized pruritus, rash, facial
swelling) in 4 vaccinees, none serious or early-onset.
Two vaccinees developed neurologic symptoms, with
pain or paresthesia radiating down the injected arm.
Both instances began within hours after vaccination,
lasted several days, were rated severe but resolved fully. Figure 1. Summary of participation in crossover protocol. Comment: one subject
withdrew before the initial injection, a second withdrew shortly after the initial
Other adverse events after placebo injection included injection (placebo, with code break) and a third withdrew prior to the second injec-
a vasovagal reaction (1), cardiac palpitations (1) and tion. Two subjects opted out of visit 3.
hyperventilation syndrome (1), all beginning within
minutes after the injection. Only one subject was hos-
pitalized during the study, after a motor vehicle accident. 37 (71.2%) retained titers ≥ 40, the GMT having declined 82%
Despite treatment blinding, 75% of subjects correctly guessed to 52.6 (95% CI 39.3, 70.3). After TIV, all but one had titers ≥
when they had received TIV, based on Day 7 interviews. A pre- 40 and GMT was 180.4 (95% CI 139, 235), a 3.4-fold increase.
liminary report of blinded safety data after first injections was Responses to A/H3N2 and B components of the vaccine are
presented to the DSMB encompassing all Day 1 and most Day included in Table 4. An association was detected between base-
7 observations. So few severe adverse events had occurred that line titers of antibody to A/H1N1pdm09 and post-immunization
the DSMB approved continuing as planned. A detailed safety myalgia. Subjects with baseline titers ≤ 20 were less likely to
report was presented to the Public Health Agency of Canada 32 d experience myalgia (25/137, 18%) than those with titers ≥ 80
after study commencement (September 10, 2010) and was widely (31/106, 29%, p < 0.05, odds ratio 0.54, 95% CI 0.298, 0.996).
shared with provincial and territorial immunization program Baseline titers of antibody to H3N2 and B viruses did not show a
administrators before public programs began. significant association with myalgia (data not shown). No associ-
Immunogenicity data. At study entry, HAI antibody to ations were detected between baseline H1N1 antibody titers and
H1N1pdm09 was detectable in most subjects (Table 4) with half other general symptoms, perhaps because of their infrequency, or
having titers ≥ 40. Age influenced the frequency of residual titers with injection site pain or erythema (data not shown).
≥ 40, which ranged from 73.4% (95% CI 60.9, 83.7) among
20–29 y olds to 42.5% (95% CI 31.5, 57.5) among 40–49 y olds Discussion
(p = 0.0004, Cochran-Armitage trend test, 2-sided). Following
vaccination (Table 4), almost all subjects developed titers ≥ 40 This study provided timely assurance that re-exposure of
and the GMT increased 4.4 fold. A subset of 52 subjects had Canadian adults to H1N1pdm09 antigen contained in TIV for
participated in a trial of AS03-adjuvanted vaccine in 2009.4 One 2010–11 would be safe, following prior vaccination with a novel,
month after the pandemic vaccine, 51 (98.1%) had titers ≥ 40, adjuvanted pandemic vaccine. The observed rates and severity
with a GMT of 285.7 (95% CI 208, 391). Nine months later, of common adverse effects were within the ranges previously

138 Human Vaccines & Immunotherapeutics Volume 9 Issue 1

 Table 2. Rates of general symptoms reported by study participants during days 0–6 after masked TIV vaccine or saline placebo injections, including
vaccine-attributable rates (as rate difference)
Symptom Following vaccine (%) Following placebo (%) Rate difference (95% CI) p value
Myalgia
Any 81/323 (25.1) 21/325 (6.5) 18.6% (13.2, 24.1) < 0.001
Mild 67 (20.7) 18 (5.5) 15.2% (10.1, 20.2) < 0.001
Moderate 11 (3.4) 3 (0.9) 2.5% (0.24, 4.7) 0.03
Severe 3 (0.9) 0 0.9% (-0.12, 2.0) 0.12

Tiredness
Any 66/323 (20.4) 34/325 (10.5) 10.0% (4.5, 15.5) < 0.001
Mild 47 (14.6) 25 (7.7) 6.9% (2.0, 11.7) 0.006
Moderate 14 (4.3) 9 (2.8) 1.6% (-1.3, 4.4) 0.298
Severe 5 (1.5) 0 1.5% (0.2, 2.9) 0.03

Headache
Any 67/323 (20.7) 40/325 (12.3) 8.4% (2.7, 14.1) 0.004
Mild 55 (17.0) 31 (9.5) 7.5% (2.3, 12.7) 0.005
Moderate 10 (3.1) 9 (2.8) 0.3% (-2.3, 2.9) 0.821
Severe 2 (0.6) 0 0.6% (-0.2, 1.5) 0.248

Malaise/Feeling unwell
Any 36/323 (11.1) 21/325 (6.5) 4.7% (0.3, 9.0) 0.038
Mild 26 (8.1) 15 (4.6) 3.4% (-0.3, 7.2) 0.078
Moderate 7 (2.2) 5 (1.5) 0.6% (-1.5, 2.7) 0.577
Severe 3 (0.9) 1 (0.3) 0.6% (-0.6, 1.8) 0.372

Arthralgia
Any 22/323 (6.8) 9/325 (2.8) 4.0% (0.8, 7.3) 0.017
Mild 18 (5.6) 7 (2.2) 3.4% (0.5, 6.4) 0.026
Moderate 2 (0.6) 2 (0.6) 0.0% (-1.2, 1.2) 1
Severe 2 (0.6) 0 0.6% (-0.2, 1.5) 0.248
Sleep disturbance 25/323 (7.7) 12/325 (3.7) 4.0% (0.5, 7.6) 0.028
Diarrhea 19/323 (5.9) 6/325 (1.8) 4.0% (1.1, 7.0) 0.008
Fever 2/322 (0.6) 2/322 (0.6) 0 (-1.2, 1.2) 1
Nausea 10/323 (3.1) 6/325 (1.8) 1.3% (-1.1, 3.6) 0.325
Vomiting 2/323 (0.6) 1/325 (0.3) 0.3% (-0.7, 1.4) 0.623

described for similar TIV vaccines.7,14,15 However, myalgia was of H3N2 and B antibodies raises the possibility that the myalgia
reported more frequently in this study than others assessing the reported by our subjects was triggered by elements of the immune
same product,14,15 including a similar placebo-controlled, cross- response specific to the H1N1pdm09 antigen itself, the adjuvant
over study in 2001–200214 in which the vaccine-attributable rate or the combination. The strong association between myalgia and
of myalgia was 2.6% (95% CI 0.4, 4.8) compared with 18.6% injection site pain suggests that whatever immune mechanisms
(95% CI 13.2, 24.1) in the present study. Peak rates of myalgia in were involved in the local inflammatory process, they generated
the current study were as high as 28% in subjects with injection cytokines with this adverse effect, such as interferon. However,
site pain and 29% in those with baseline H1N1pdm09 HAI titers confirmation of this hypothesis would have required inclusion of
≥ 80. However, most instances were mild/moderate and short- a control group of adults not previously exposed to the adjuvanted
lived. To our knowledge, such a titer threshold effect for an adverse vaccine. In a study of UK children16 similarly revaccinated with
event following influenza vaccination has not previously been TIV in 2010 after receiving 2 doses of AS03-adjuvanted or whole
reported. The lack of significant association with baseline titers virion H1N1pdm09 vaccine a year earlier, injection site redness

www.landesbioscience.com Human Vaccines & Immunotherapeutics 139

 Table 3. Participants with multiple new respiratory symptoms within one day after vaccine or placebo administration
Difficulty
Subject Red eyes Sore throat Hoarseness Coughing Chest tightness Difficulty breathing Wheezing Facial swelling
swallowing
After vaccine (n = 11)
V1 0 0 0 Mild Mild Mild Mild 0 0
V2 Mild 0 Mild Mild Moderate Moderate 0 0 0
V3 0 Mild Mild 0 0 0 0 0 0
V4 0 0 Mild Mild Mild 0 0 0 0
V5 0 Mild Mild 0 0 0 0 0 0
V6 0 0 0 Mild 0 0 Mild 0 0
V7 0 Mild Mild Mild 0 0 0 0 0
V8 Mild 0 0 0 0 0 0 0 Moderate
V9 0 Moderate 0 Mild 0 0 0 0 Moderate
V10 0 0 0 Moderate 0 Mild 0 0 0
V11 0 0 Mild Mild 0 0 0 0 0
After placebo (n = 3)
P1 Mild Moderate Moderate 0 0 0 0 Moderate 0
P2 0 Moderate Moderate Moderate 0 0 0 0 Mild
P3 0 0 0 Mild Mild 0 Mild 0 0

and severe local reactions were more frequent in children < 5 y Table 4. HAI antibody responses to immunization with 2010 TIV vaccine,
of age given the adjuvanted vaccine, supporting the possibility of in adults previously given adjuvanted H1N1 2009 pandemic vaccine
unique effects after the latter vaccine. Two studies of Canadian A/H3N2
Parameter A/H1N1/2009 B (Brisbane)
children17,18 re-exposed to H1N1pdm09 antigen in 2010 after (Perth)
receiving AS03-adjuvanted vaccine in 2009 reported no unusual A. Baseline (n = 325)
increase in adverse effects after vaccination but also lacked a com- HAI ≥ 10 303 (93.2%) 167 (51.4%) 323 (99.4%)
parison group not primed with adjuvanted vaccine.
HAI ≥ 40 176 (54.2%) 60 (18.5%) 272 (83.7%)
Our screening for possible unusual adverse effects of re-
(95% CI) (48.6, 59.7) (14.4, 23.1) (79.2, 87.5)
vaccination with H1N1/2009 antigen included monitoring for
symptoms of oculorespiratory syndrome (ORS).19-22 We expected GMT 37.4 13.1 99.4
viral respiratory infections to be infrequent during a mid-sum- (95% CI) (32.8, 42.6) (11.9, 14.4) (88.0, 112)
mer study. ORS was described in 2000 as an adverse effect of B. Post-immunization
a Canadian-manufactured TIV (Fluviral®, Shire Biologics) for (n = 321)
the 2000–2001 season.19,20,22 The syndrome was defined as onset HAI ≥ 40 308 (96.0%) 230 (71.7%) 317 (98.8%)
within 2–24 h after vaccination of any of bilateral red eyes, facial (95% CI) (93.2, 97.8) (66.4, 76.5) (96.8, 99.7)
swelling, cough, hoarseness, sore throat, difficulty swallowing,
GMT 167.4 61.3 225.5
wheezing, chest tightness or difficulty breathing, not obviously
(95% CI) (148.7, 188.5) (53.2, 70.6) (200.2, 254.1)
associated with an allergic reaction or respiratory infection. The
overall ORS rate in 2000 was estimated at 3.4% of vaccinated GM fold rise 4.4 4.7 2.28
adults, ranging as high as 16% in women 40–59 y old.22 Between (95% CI) (3.9, 5.0) (4.1, 5.3) (2.0, 2.6)
10–24% of individuals with ORS consulted a health care pro- Seroconversion 191 (59.5%) 180 (56.1%) 86 (26.5%)
vider.19,20 A re-formulated product for 2001–2002 (using a sec- (95% CI) (53.9, 64.9) (50.5, 61.6) (21.7, 31.6)
ond virus splitting detergent) was shown to cause mild ORS at
a rate of 2.9% among vaccinated adults.14 In the present study
we applied a more stringent case definition than used for sur- detected some infrequent events such as generalized pruritus and
veillance of case reports, given the high rate at which vaccinees limb pain/paresthesia it was not possible to predict whether they
and controls described new-onset, single, mild respiratory symp- were unique to the study population or would be seen as infre-
toms. With a requirement for two or more eligible symptoms, quent events in the mass programs. Because of their timing and
the observed rate of ORS-like symptoms after vaccination was evolution, the neurological events were likely related to the “act of
3.4%, with a vaccine-attributable rate of 2.5%. All instances vaccination” rather than the vaccine components themselves, as
were mild to moderate, with none requiring medical attention. has been previously reported in other immunization programs.23
On this basis it seemed unlikely that ORS would occur with Ferguson et al.2 recently reported that 83% of adults 18–65 y
increased frequency or severity in public programs. While we of age given one dose of AS03-adjuvanted H1N1pdm09 vaccine

140 Human Vaccines & Immunotherapeutics Volume 9 Issue 1

in a prospective study had HAI titers ≥ 40 six months after the Ottawa, Calgary and Vancouver. A number of strategies were
vaccination. In the present study, 9–10 mo after such vaccination employed to accelerate enrollment and the safety evaluation,
in field conditions, 54% of adults 19–59 y old still had titers ≥ 40. which have been described separately.13
Both studies showed greater titer declines in older than younger Eligible subjects were 20–59 y of age, in generally good health.
adults, as was also described after administration of two doses of Stable chronic health conditions without immunocompromise
MF59-adjuvanted pandemic vaccine.24 This TIV-like decay rate7 were acceptable. Subjects had to have received AS03-adjuvanted
likely reflected the low dose of antigen in the AS03-adjuvanted A/H1N1pdm09 influenza vaccine (Arepanrix®) in late 2009.
vaccine, intended as a dose-sparing strategy. Rapid loss of pro- Exclusion criteria included: allergy to egg or other vaccine con-
tection after this dosage could have been problematic had the stituent; bleeding disorder; pregnancy; immune compromise
pandemic continued so the optimal balance between dose mini- from medication or illness; recent blood product infusion; or
mization and duration of protection warrants further study. prior receipt of a 2010–2011 influenza vaccine. Informed consent
Effective memory responses were evident after the low dose of was obtained from each subject at study entry. The study was
antigen as all subjects boosted well with TIV 9–10 mo later, with conducted in accordance with the Code of Ethics of the World
almost 100% developing titers ≥ 40. However, the post-vaccina- Medical Association (Declaration of Helsinki) and was approved
tion GMT of H1N1pdm09 antibodies was just 2.7-fold higher by the research ethics board of each participating institution. The
than the GMT of H3N2 antibodies, while the GM fold-rise and ClinicalTrials.gov registry identifier was NCT01140009.
seroconversion rates were nearly identical with the two influenza A web-based central randomization method (Daciforms®)
A strains, speaking against any long-lived benefit in terms of HAI was used to assign subjects (1:1) to receive influenza vaccine
responses after exposure to adjuvanted vaccine. or placebo at the first visit. Randomization assignments were
A strength of this study was the randomized, blinded, placebo- computer-generated (Proc Plan in SAS, SAS Institute) in bal-
controlled design. Our experiences with anxiety-related events anced blocks of four and stratified by sex and age (20–39 and
after placebo injections and with closely-scrutinized respiratory 40–59 y). Participants were given the opposite assignment at
symptoms underscore the value of placebo-controlled observa- Visit 2, ten days later, with masking maintained. The 10-d inter-
tions when assessing vaccine safety. The crossover trial design val was selected to allow resolution of any adverse effects after
provides an efficient means of obtaining placebo-controlled vaccination.
observations in perfectly matched groups. Unfortunately, reacto- The study vaccine (Fluviral®) was an egg-derived, formalin-
genicity of the vaccine and placebo differed sufficiently to enable inactivated, detergent-split preparation, containing thimerosal as
75% of participants to correctly guess when they had been given preservative, formulated for the 2010–11 Northern Hemisphere
TIV, potentially diminishing their objectivity. Recruiting mainly winter season. It contained 15 μg of hemagglutinin from each of
healthcare workers may have increased symptom ascertainment A/California/7/2009 (H1N1v)-like, A/Perth/16/2009 (H3N2)-
bias but facilitated high protocol compliance. Participants had like and B/Brisbane/60/2008-like viruses, per 0.5 mL dose. A
a high rate of past TIV vaccinations, residual effects of which single lot was used (#AFLLA574AB), obtained prior to commer-
may have narrowed the differences between H1N1pdm09 and cial sale authorization with approval of the Biologic and Genetic
H3N2 responses. Other limitations included our inability to Therapies Directorate of Health Canada. The placebo was nor-
include participants known to have had natural H1N1pdm09 mal saline for injection, locally sourced. Injectables were pre-
infection, to determine if they responded differently to the subse- pared out of sight of subjects; vaccine and placebo were identical
quent seasonal vaccination. As only single pandemic and seasonal in appearance. Both were stored at 2–8°C and injected into the
influenza vaccines were assessed, our observations may not reflect deltoid muscle using 25 gauge needles, one inch (25 mm) long.
antibody decline after other H1N1pdm09 vaccines or the effects Only the nurse who administered injections had access to the
of re-exposure to other seasonal influenza vaccines for 2010– treatment information; all other study staff remained blinded,
2011. In Dutch adults24 given two doses of MF59-adjuvanted including safety interviewers.
H1N1pdm09 vaccine, antibody decline was remarkably simi- Participants were observed for at least 15 min after each
lar to our observations, suggesting that the shared components injection. During this time they were given instructions about
(squalene and H1N1 antigen) determined the pattern of decline. using a daily symptom diary (supplied) to record any solicited
Second doses of the MF59-adjuvanted vaccine increased titers or unsolicited local, respiratory or general symptoms, including
significantly only in persons over age 50 so the dosing regimens fever, for 6 d after each injection. An electronic thermometer was
were not too dissimilar between that study and the present one. supplied to measure oral temperature, as was a device to mea-
sure injection site redness and swelling. Subjects were contacted
Methods and Participants by telephone one and seven days after each injection to review
and record any symptoms. Day 7 interviews included a ques-
In this prospective, randomized, evaluator-blinded, crossover tion about which injection (vaccine or placebo) subjects thought
trial, each subject received vaccine and placebo, in random that they had received, with blinding maintained. The purpose
sequence. The study was conducted at 5 academic centers across of the initial telephone contact was early detection of any severe
Canada belonging to the PHAC/CIHR Influenza Research symptoms. Health care utilization and symptom resolution were
Network (PCIRN),4,5 during August and September, 2010. reviewed during each clinic visit and throughout the period of
Participating centers were located in Quebec City, Montreal, study participation. Safety information was entered into a secure,

www.landesbioscience.com Human Vaccines & Immunotherapeutics 141

web-based reporting system (Daciforms®) within 24 h after each mo later. While the low-dose formulation was intended to be
encounter and reviewed centrally for completeness. A study stat- dose-sparing, the short duration of protection could have been
istician tallied severe adverse event numbers daily and cumu- problematic had the pandemic been more prolonged. However,
latively, without breaking the assignment code, to monitor for individuals given a subsequent non-adjuvanted dose of TIV
any increased vaccine reactogenicity. A Data Safety Monitoring containing the H1N1pdm09 antigen boosted well, reflecting
Board (DSMB) reviewed severe adverse event rates after the first excellent immune memory. The second vaccination with the
round of injections, before the second round was undertaken. H1N1pdm09 antigen was generally well tolerated and did not
Blood samples (8–10 mL) were obtained from subjects at generate unusually high anti-H1N1 titers. These observations
study entry and 21–30 d after the TIV vaccination. To determine may prove useful in designing dosing regimens for future adju-
the correct sampling date it was necessary to un-blind the group vanted influenza vaccines.
assignment after safety assessments were completed 7 d after the
second injection. Blood samples were processed promptly and Disclosure of Potential Conflicts of Interest
sera were stored at -20°C or colder pending testing at the national No potential conflicts of interest were disclosed.
reference laboratory. Serum pairs were tested concurrently, in
duplicate, using hemagglutination inhibition (HAI) assays for Financial Disclosure Statement
each vaccine strain per WHO methods.25 Live viruses were used This study was funded entirely by a grant to the Public Health
for influenza A assays and ether-treated viruses for influenza B Agency of Canada/Canadian Institutes of Health Research
assays. Minimum sensitivity was a titer of 1:10. Results of dupli- Influenza Research Network (PCIRN). GSK Canada supplied
cate tests on samples were expressed as the geometric mean titer the influenza vaccine (Fluviral) for this trial and contributed
(GMT). an unrestricted grant toward a similar, concurrent PCIRN trial
Adverse event rates were determined daily and cumulatively in children (ref. 18), with investigators that included D.W.S.,
for days 0–6 after each injection. The cumulative rate of indi- O.G.V., B.J.W., Y.L. and S.A.H. D.W.S. was the principal investi-
vidual symptoms after placebo was subtracted from the rate after gator and has received previous study funds and consulting hono-
vaccination to determine the vaccine-attributable rate. Symptom raria from GSK, sanofi, Novartis and Pfizer.
severity ratings were provided by subjects according to criteria
printed on the diary forms. Severe symptom rates were the pri- Trademark Statement
mary safety outcome, defined as fever > 40.0°C, injection site Arepanrix and Fluviral are trademarks of the GlaxoSmithKline
redness or swelling ≥ 100 mm diameter or any symptom that group of companies.
precluded normal daily activities or prompted medical attention.
Serologic responses were analyzed according to standard inter- Acknowledgments
national (EMEA/CHMP)26 criteria for adults < 60 y old given All named authors participated in the implementation of the
seasonal influenza vaccine. Seroprotection was considered the study, including contributions toward the study design and gath-
primary serologic outcome measure, defined as an HAI titer ≥ ering and interpretation of the data. All authors were involved in
40. Geometric mean titers were calculated, with any test-nega- drafting the article and approved the final draft.
tive sample assigned to value of 5. Geometric mean fold rise was Contributing PCIRN Influenza Research Network investi-
calculated as the within-subjects ratios of the post-vaccination gators included Gaston De Serres MD, PhD (Institut national
HAI titer to the baseline HAI titer. The seroconversion rate was de santé publique du Québec, Laval University), James Kellner
defined as either a 4-fold or greater titer rise or conversion from a MD (University of Calgary, Alberta Children’s Hospital), Simon
negative baseline titer to one ≥ 40. Dobson MD (University of British Columbia, BC Children’s
A sample size of 300 evaluable subjects was desired to permit Hospital), Nathalie Bastien PhD (National Microbiology
detection of severe or unusual adverse events at rates as low as 1% Laboratory) and Barbara Law MD (Public Health Agency of
with 90% probability. Event rate differences after vaccine and Canada).
placebo ≥ 6% could be detected with 80% probability (α = 0.05) We thank the DSMB members: Gaston De Serres MD (chair),
with 300 observations after each treatment, using Fisher’s exact Harold Rode MD, Robert Pless MD (Public Health Agency of
test. To allow for drop-outs, the intended enrollment was 320 Canada) and Jean Luc Grenier MD (Direction de la santé pub-
subjects, 64 per center. lique des Laurentides).
We greatly appreciated the skilled assistance of the project
Conclusion manager (Carol LaJeunesse), data manager (Kim Marty), stat-
istician (Shuyu Fan) and the coordinators and staff at each par-
The data from this study showed that adults given a low-dose, ticipating center. Presented in brief at the Fourteenth Annual
ASO3-adjuvanted H1N1pdm09 vaccine in 2009 had substan- Conference on Vaccine Research in Baltimore, MD, on May
tially reduced rates of seroprotection (HAI titers ≥ 40) 9–10 16–18, 2011 (Abstract S18).

142 Human Vaccines & Immunotherapeutics Volume 9 Issue 1

 11. Levine L, Edsall G. Tetanus toxoid: what deter- 19. De Serres G, Grenier JL, Toth E, Ménard S, Roussel
References
mines reaction proneness? J Infect Dis 1981; R, Tremblay M, et al. The clinical spectrum of the
1. Girard MP, Tam JS, Assossou OM, Kieny MP. The 144:376; PMID:7288216; http://dx.doi.org/10.1093/ oculo-respiratory syndrome after influenza vaccination.
2009 A (H1N1) influenza virus pandemic: A review. infdis/144.4.376. Vaccine 2003; 21:2354-61; PMID:12744866; http://
Vaccine 2010; 28:4895-902; PMID:20553769; http:// 12. Armstrong PK, Dowse GK, Effler PV, Carcione D, dx.doi.org/10.1016/S0264-410X(03)00094-X.
dx.doi.org/10.1016/j.vaccine.2010.05.031. Blyth CC, Richmond PC, et al. Epidemiological 20. Skowronski DM, Strauss B, De Serres G, MacDonald
2. Ferguson M, Risi G, Davis M, Sheldon E, Baron M, study of severe febrile reactions in young children in D, Marion SA, Naus M, et al. Oculo-respiratory
Li P, et al. Safety and long-term humoral immune Western Australia caused by a 2010 trivalent inacti- syndrome: a new influenza vaccine-associated
response in adults after vaccination with an H1N1 vated influenza vaccine. BMJ Open 2011; 1:e000016; adverse event? Clin Infect Dis 2003; 36:705-13;
2009 pandemic influenza vaccine with or without PMID:22021725; http://dx.doi.org/10.1136/bmjo- PMID:12627354; http://dx.doi.org/10.1086/367667.
AS03 adjuvant. J Infect Dis 2012; 205:733-44; pen-2010-000016. 21. De Serres G, Toth E, Ménard S, Grenier JL, Roussel
PMID:22315336; http://dx.doi.org/10.1093/infdis/ 13. Scheifele DW, Marty K, LaJeunesse C, Fan SY, R, Tremblay M, et al. Oculo-respiratory syndrome
jir641. Bjornson G, Langley JM, et al. Strategies for suc- after influenza vaccination: trends over four influenza
3. Roman F, Vaman T, Gerlach B, Markendorf A, Gillard cessful rapid trials of influenza vaccine. Clin Trials seasons. Vaccine 2005; 23:3726-32; PMID:15882534;
P, Devaster JM. Immunogenicity and safety in adults of 2011; 8:699-704; PMID:21900340; http://dx.doi. http://dx.doi.org/10.1016/j.vaccine.2005.01.154.
one dose of influenza A H1N1v 2009 vaccine formu- org/10.1177/1740774511419868. 22. Boulianne N, De Serres G, Duval B, Shadmani R,
lated with and without AS03A-adjuvant: preliminary 14. Scheifele DW, Duval B, Russell ML, Warrington Rochette L. Clinical manifestations and incidence of
report of an observer-blind, randomised trial. Vaccine R, DeSerres G, Skowronski DM, et al. Ocular and oculo-respiratory syndrome following influenza vacci-
2010; 28:1740-5; PMID:20034605; http://dx.doi. respiratory symptoms attributable to inactivated split nation--Quebec, 2000. Can Commun Dis Rep 2001;
org/10.1016/j.vaccine.2009.12.014. influenza vaccine: evidence from a controlled trial 27:85-90; PMID:11396047.
4. Scheifele DW, Ward BJ, Dionne M, Vanderkooi O, involving adults. Clin Infect Dis 2003; 36:850-7; 23. Sever JL, Brenner AI, Gale AD, Lyle JM, Moulton LH,
Loeb M, Coleman BL, et al. Compatibility of AS03- PMID:12652385; http://dx.doi.org/10.1086/368189. West DJ; Anthrax Vaccine Export Committee. Safety
adjuvanted A/H1N1pdm09 and seasonal trivalent 15. Jackson LA, Gaglani MJ, Keyserling HL, Balser J, of anthrax vaccine: a review by the Anthrax Vaccine
influenza vaccines in adults: results of a randomized, Bouveret N, Fries L, et al. Safety, efficacy, and immuno- Expert Committee (AVEC) of adverse events report-
controlled trial. Vaccine, In press. genicity of an inactivated influenza vaccine in healthy ed to the Vaccine Adverse Event Reporting System
5. Rubinstein E, Predy G, Sauvé L, Hammond GW, Aoki adults: a randomized, placebo-controlled trial over (VAERS). Pharmacoepidemiol Drug Saf 2002; 11:189-
F, Sikora C, et al. The responses of Aboriginal Canadians two influenza seasons. BMC Infect Dis 2010; 10:71; 202; PMID:12051118; http://dx.doi.org/10.1002/
to adjuvanted pandemic (H1N1) 2009 influenza vac- PMID:20236548; http://dx.doi.org/10.1186/1471- pds.712.
cine. CMAJ 2011; 183:E1033-7; PMID:21788422; 2334-10-71. 24. Huijskens E, Rossen J, Mulder P, van Beek R, van Vugt
http://dx.doi.org/10.1503/cmaj.110196. 16. Walker WT, de Whalley P, Andrews N, Oeser C, Casey H, Verbakel J, et al. Immunogenicity, boostability, and
6. Canadian community health survey: H1N1 vac- M, Michaelis L, et al. H1N1 antibody persistence sustainability of the immune response after vaccination
cinations. Ottawa (ON): Statistics Canada; 2010. 1 year after immunization with an adjuvanted or against Influenza A virus (H1N1) 2009 in a healthy
Available: www.statcan.gc.ca/daily-quotidien/100719/ whole-virion pandemic vaccine and immunogenic- population. Clin Vaccine Immunol 2011; 18:1401-
dq100719b-eng.htm (accessed 2011 Sept 12). ity and reactogenicity of subsequent seasonal influ- 5; PMID:21795459; http://dx.doi.org/10.1128/
7. Beyer WEP, Nauta JJP, Palache AM, Giezeman KM, enza vaccine: a multicenter follow-on study. Clin Infect CVI.05046-11.
Osterhaus ADME. Immunogenicity and safety of Dis 2012; 54:661-9; PMID:22267719; http://dx.doi. 25. World Health Organization. WHO Manual on Animal
inactivated influenza vaccines in primed populations: a org/10.1093/cid/cir905. Influenza Diagnosis and Surveillance, 2002. Available
systematic literature review and meta-analysis. Vaccine 17. Gilca V, De Serres G, Hamelin ME, Boivin G, Ouakki at: www.who.int/vaccine research/diseases/influenza/
2011; 29:5785-92; PMID:21624411; http://dx.doi. M, Boulianne N, et al. Antibody persistence and WHO manual on animal-diagnosis and surveillance
org/10.1016/j.vaccine.2011.05.040. response to 2010-2011 trivalent influenza vaccine one 2002 5.pdf [accessed February 15, 2012]
8. World Health Organization. Recommended virus- year after a single dose of 2009 AS03-adjuvanted pan- 26. Wood JM, Newman RW, Ploss K. The use of cor-
es for influenza vaccines for use in the 2010-2011 demic H1N1 vaccine in children. Vaccine 2011; 30:35- relates of immunity in European Union licensing of
northern hemisphere influenza season. Available: 41; PMID:22063386; http://dx.doi.org/10.1016/j.vac- influenza vaccines. Dev Biol (Basel) 2003; 115:9-16;
www.who.int/influenza/vaccines/recommendations/ cine.2011.10.062. PMID:15088770.
recommendation2010_11north/en/index/html 18. Langley JM, Scheifele DW, Quach C, Vanderkooi O,
(accessed 2011 Sept 12). Ward B, McNeil S, et al. Safety and immunogenic-
9. Scheifele DW, Ochnio JJ, Halperin SA. Cellular ity of 2010-2011 H1N1 2009-containing trivalent
immunity as a potential cause of local reactions to inactivated influenza vaccine in children 12-59 months
booster vaccination with diphtheria and tetanus tox- of age previously given AS03-adjuvanted H1N1 2009
oids and acellular pertussis antigens. Pediatr Infect Dis pandemic vaccine: a PHAC/CIHR Influenza Research
J 2009; 28:985-9; PMID:19755930; http://dx.doi. Network (PCIRN) study. Vaccine 2012; 23:3389-94;
org/10.1097/INF.0b013e3181a9cc2a. http://dx.doi.org/10.1016/j.vaccine.2012.03.046.
10. Rowe J, Yerkovich ST, Richmond P, Suriyaarachchi
D, Fisher E, Feddema L, et al. Th2-associated local
reactions to the acellular diphtheria-tetanus-pertussis
vaccine in 4- to 6-year-old children. Infect Immun
2005; 73:8130-5; PMID:16299307; http://dx.doi.
org/10.1128/IAI.73.12.8130-8135.2005.

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