Low 2012–13 Influenza Vaccine Effectiveness Associated
with Mutation in the Egg-Adapted H3N2 Vaccine Strain
Not Antigenic Drift in Circulating Viruses
Danuta M. Skowronski1,2*, Naveed Z. Janjua2,3, Gaston De Serres4,5, Suzana Sabaiduc1, Alireza Eshaghi6,
James A. Dickinson7, Kevin Fonseca8,9, Anne-Luise Winter10, Jonathan B. Gubbay11,12,13, Mel Krajden1,3,
Martin Petric1,3, Hugues Charest14,15, Nathalie Bastien16, Trijntje L. Kwindt2, Salaheddin M. Mahmud17,
Paul Van Caeseele18,19, Yan Li16,19
1 Communicable Disease Prevention and Control Service, British Columbia Centre for Disease Control, Vancouver, British Columbia, Canada, 2 School of Population and
Public Health, University of British Columbia, Vancouver, British Columbia, Canada, 3 Clinical Prevention Services, British Columbia Centre for Disease Control, Vancouver,
British Columbia, Canada, 4 Department of Biological and Occupational Risks, Institut National de Santé Publique du Québec, Québec (Québec), Canada, 5 Department of
Social and Preventive Medicine, Laval University, Québec (Québec), Canada, 6 Department of Molecular Research, Public Health Ontario, Toronto, Ontario, Canada,
7 Family Medicine and Community Health Sciences, University of Calgary, Calgary, Alberta, Canada, 8 Department of Virology, Provincial Laboratory of Public Health,
Calgary, Alberta, Canada, 9 Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada, 10 Communicable Disease
Prevention and Control, Public Health Ontario, Toronto, Ontario, Canada, 11 Department of Microbiology, Public Health Ontario, Toronto, Ontario, Canada,
12 Department of Laboratory Medicine and Pathobiology and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada, 13 Department of Paediatrics,
The Hospital for Sick Children, Toronto, Ontario, Canada, 14 Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-deBellevue, Québec, Canada, 15 Département De Microbiologie, Infectiologie et Immunologie, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada,
16 Influenza and Respiratory Virus Section, National Microbiology Laboratory, Winnipeg, Manitoba, Canada, 17 Community Health Sciences and Pharmacy, University of
Manitoba, Winnipeg, Manitoba, Canada, 18 Cadham Provincial Laboratory, Manitoba Health, Winnipeg, Manitoba, Canada, 19 Department of Medical Microbiology,
University of Manitoba, Winnipeg, Manitoba, Canada
Abstract
Background: Influenza vaccine effectiveness (VE) is generally interpreted in the context of vaccine match/mismatch to
circulating strains with evolutionary drift in the latter invoked to explain reduced protection. During the 2012–13 season,
however, detailed genotypic and phenotypic characterization shows that low VE was instead related to mutations in the
egg-adapted H3N2 vaccine strain rather than antigenic drift in circulating viruses.
Methods/Findings: Component-specific VE against medically-attended, PCR-confirmed influenza was estimated in Canada
by test-negative case-control design. Influenza A viruses were characterized genotypically by amino acid (AA) sequencing of
established haemagglutinin (HA) antigenic sites and phenotypically through haemagglutination inhibition (HI) assay. H3N2
viruses were characterized in relation to the WHO-recommended, cell-passaged vaccine prototype (A/Victoria/361/2011) as
well as the egg-adapted strain as per actually used in vaccine production. Among the total of 1501 participants, influenza
virus was detected in 652 (43%). Nearly two-thirds of viruses typed/subtyped were A(H3N2) (394/626; 63%); the remainder
were A(H1N1)pdm09 (79/626; 13%), B/Yamagata (98/626; 16%) or B/Victoria (54/626; 9%). Suboptimal VE of 50% (95%CI: 33–
63%) overall was driven by predominant H3N2 activity for which VE was 41% (95%CI: 17–59%). All H3N2 field isolates were
HI-characterized as well-matched to the WHO-recommended A/Victoria/361/2011 prototype whereas all but one were
antigenically distinct from the egg-adapted strain as per actually used in vaccine production. The egg-adapted strain was
itself antigenically distinct from the WHO-recommended prototype, and bore three AA mutations at antigenic sites B
[H156Q, G186V] and D [S219Y]. Conversely, circulating viruses were identical to the WHO-recommended prototype at these
positions with other genetic variation that did not affect antigenicity. VE was 59% (95%CI:16–80%) against A(H1N1)pdm09,
67% (95%CI: 30–85%) against B/Yamagata (vaccine-lineage) and 75% (95%CI: 29–91%) against B/Victoria (non-vaccinelineage) viruses.
Conclusions: These findings underscore the need to monitor vaccine viruses as well as circulating strains to explain vaccine
performance. Evolutionary drift in circulating viruses cannot be regulated, but influential mutations introduced as part of
egg-based vaccine production may be amenable to improvements.
Citation: Skowronski DM, Janjua NZ, De Serres G, Sabaiduc S, Eshaghi A, et al. (2014) Low 2012–13 Influenza Vaccine Effectiveness Associated with Mutation in
the Egg-Adapted H3N2 Vaccine Strain Not Antigenic Drift in Circulating Viruses. PLoS ONE 9(3): e92153. doi:10.1371/journal.pone.0092153
Editor: Gary P. Kobinger, Public Health Agency of Canada, Canada
Received December 29, 2013; Accepted February 17, 2014; Published March 25, 2014
Copyright: ß 2014 Skowronski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by the Canadian Institutes of Health Research (CIHR) – Institute of Infection and Immunity, grant TPA-90193 (http://www.
cihr-irsc.gc.ca/), as well as Ministries of Health and Institutes of the investigators. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. This does not alter adherence to all PLOS policies on sharing data and materials.
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2012–13 Influenza Vaccine Effectiveness
Competing Interests: Within 36 months of manuscript submission, GDS received research grants from GlaxoSmithKline (GSK) and Sanofi Pasteur for unrelated
vaccine studies and travel fee reimbursement to attend an ad hoc GSK Advisory Board, without honorarium. JBG has received research grants from GSK and
Hoffmann-LaRoche for antiviral resistance studies. MK has received research grants from Roche, Merck, Gen-Probe and Siemens. SMM has received research grants
from GSK, Sanofi Pasteur and Pfizer. SMM is a Canada Research Chair in Pharmaco-epidemiology and Vaccine Evaluation; and the Great-West Life, London Life and
Canada Life Junior Investigator of the Canadian Cancer Society [grant # 2011-700644]. SS and TLK are funded by the Canadian Institutes of Health Research Grant
(TPA-90193). The other authors declare that they have no competing interests to report. This does not alter adherence to all PLOS policies on sharing data and
materials.
* E-mail: danuta.skowronski@bccdc.ca
Epidemiologic
Introduction
A test-negative case-control design embedded within the routine
sentinel surveillance network has been used each year in Canada
since 2004 to estimate effectiveness of the annually-reformulated
trivalent influenza vaccine (TIV) [1,12–19]. Several hundred
practitioners from designated community-based sentinel sites in
the five most-populous provinces (British Columbia (BC), Alberta,
Manitoba, Ontario and Québec) contribute to annual virologic
and VE monitoring. Participating sentinel sites can offer nasal or
nasopharyngeal swabs for influenza virus testing to all patients
presenting within 7 days of influenza-like illness (ILI) onset. ILI is
defined as acute fever and cough illness with one or more of sore
throat, arthralgia, myalgia or prostration. Fever is not required for
elderly patients aged $65 years.
At the time of specimen collection, the attending practitioner
also obtains epidemiologic information directly from consenting
patients/parents/guardians using a standardized questionnaire
affixed to the laboratory requisition. Information includes date of
symptom onset, current influenza immunization status and
month/year of vaccine receipt, as well as prior TIV (2011–12,
2010–11) and 2009 monovalent A(H1N1)pdm09 vaccine receipt
[17]. Details related to special pediatric immunization dosing are
not sought. Information on comorbidity is recorded on the
questionnaire as ‘yes’, ‘no’ or ‘unknown’ to any one or more of the
chronic medical conditions defined by Canada’s National Advisory Committee on Immunization as increasing the risk of
influenza complications, without specifying the condition [20].
In Canada, as elsewhere in North America, an early and intense
epidemic peak distinguished the 2012–13 influenza season [1–5].
Influenza A/H3N2 subtype viruses predominated and were
associated with increased outbreak reports from long-term care
facilities, exceeding tallies of the prior decade in some regions
despite higher immunization coverage among residents and staff in
those settings [6,7]. Consistent with these surveillance observations, mid-season assessment of vaccine performance by the
established sentinel monitoring system in Canada showed disappointing vaccine effectiveness (VE) of 45% (95%CI: 13–66%) for
the H3N2 component [1], similarly low in the United States [8]
and Europe [9]. Although suboptimal vaccine performance has
historically been linked to evolutionary drift in circulating viruses,
H3N2 viruses in Canada and elsewhere globally were characterized throughout the epidemic as antigenically similar to the
prototype virus (A/Victoria/361/2011) recommended as 2012–13
vaccine component by the World Health Organization (WHO)
[2–5,10].
To understand low VE despite reports of vaccine match, we
conducted further epidemiologic and laboratory investigations in
end-of-season analyses. With additional participants and contributing viruses, we estimated VE against circulating strains
belonging to both influenza A subtypes and B lineages accompanied by their in-depth genotypic and phenotypic characterization
in relation to vaccine components. Specifically, vaccine-virus
relatedness was assessed genotypically by determining the amino
acid (AA) sequence of established haemagglutinin (HA) antigenic
sites and phenotypically through the haemagglutination inhibition
(HI) assay. For H3N2, virus characterization was in relation to the
A/Victoria/361/2011 prototype strain recommended by the
WHO [10], as well as the egg-adapted high growth reassortant
strain as per that actually used by manufacturers in vaccine
production (hereafter ‘‘IVR-165’’) [11]. We show that suboptimal
VE for the H3N2 component during the 2012–13 season was
related to mutations in the egg-adapted IVR-165 vaccine strain,
rather than antigenic drift in circulating viruses.
Immunization
Immunized participants primarily receive vaccine during the
regular autumn immunization campaign. Influenza vaccine is
provided free of charge to all citizens $6 months old in Alberta,
Manitoba and Ontario. In BC and Québec vaccine is provided
free of charge to high-risk individuals and their close contacts or
caregivers [20]; others are also encouraged to receive vaccine but
must purchase it. For the 2012–13 season, 70% of the national
contractual volume of publicly-funded non-adjuvanted, inactivated TIV that was administered was split virus formulation and the
rest was subunit. Live attenuated influenza vaccine was also
available for those 2–59 years old, but publicly funded only in the
participating provinces of Alberta and Québec. An adjuvanted
subunit TIV formulation was also available for the elderly but used
only in the participating provinces of BC and Ontario.
For the northern hemisphere’s 2012–13 TIV, two of three
components were changed from the prior season [10]. The WHO
recommended a strain-level change for the H3N2 component to
include A/Victoria/361/2011-like prototype virus and a lineagelevel change to include B/Wisconsin/1/2010(Yamagata-lineage)like virus. The A/California/7/2009(H1N1)-like virus (hereafter
A(H1N1)pdm09) was retained unchanged since 2009 [10]. Manufacturers substituted the egg-adapted high growth reassortant
strains A/Victoria/361/2011(H3N2)-IVR-165, A/California/7/
2009(H1N1)-NYMC-X-179A (or X-181) (hereafter ‘‘X-179A’’ or
‘‘X-181’’) and B/Hubei-Wujiagang/158/2009-NYMC-BX-39 as
Methods
Ethics statement
Associated institutional ethics review boards in each contributing province approve this annual evaluation of influenza VE in
Canada based on documented oral consent, including the
Behavioural Research Ethics Board of the University of British
Columbia, the Conjoint Health Research Ethics Board of the
Calgary Health Region of Alberta Health and the University of
Calgary, the Health Research Ethics Board of the University of
Manitoba, the Health Sciences Research Ethics Board of the
University of Toronto and the University Health Network
(Ontario) and the Comité d’éthique de santé publique, Ministère
de la Santé et des Services sociaux du Québec.
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2012–13 Influenza Vaccine Effectiveness
considered antigenically-equivalent to the WHO-recommended
prototype viruses. Of the publicly supplied TIV in Canada, 70%
included A/California/7/2009-like antigen derived from X-179A
and 30% from X-181.
We also separately assessed VE in patients without comorbidity,
by age category and by prior immunization history.
Laboratory
Participant profile
Results
There were 1501 participants included in final 2012–13 VE
analysis (Figure 1). Similar to previous participation in our
sentinel network, adults 20–49 years of age comprised the greatest
proportion (680/1501; 45%) (Table 1) [1,14–19]. Overall, 16%
(107/664) of cases and 30% (263/888) of controls reported receipt
of 2012–13 TIV (p,0.01). After applying exclusion criteria related
to immunisation timing, 15% of cases and 26% of controls were
considered immunized (p,0.01) (Table 1). Only a minority of
participants reported receipt of live vaccine overall or among
children, or adjuvanted formulation for the elderly (Table 1). The
proportion of controls immunised is comparable to that of
previous VE analyses [1,14–16,18,19] and to population immunization coverage separately reported by the Canadian Community Health Survey (CCHS) (,30%) [25]. The proportion with
comorbidity (22%) was also comparable to previous seasons and to
CCHS estimates (,15–20%) (Table 1) [1,14–19,26].
The majority of those considered immunized in 2012–13 also
reported prior immunization: 83/91 (91%) cases and 180/206
(87%) controls were immunized in 2011–12 (p = 0.34); 74/85
(87%) cases and 162/199 (81%) controls were immunized in both
2011–12 and 2010–11 (p = 0.24); and 67/83 (81%) cases and 149/
189 (79%) controls received the 2009 monovalent A(H1N1)pdm09
vaccine (p = 0.72).
Specimens were tested for influenza virus at provincial public
health laboratories by real-time reverse-transcription polymerase
chain reaction (RT-PCR). All RT-PCR positive specimens were
inoculated into mammalian cell culture (Madin Darby canine
kidney (MDCK) or rhesus monkey kidney (RMK) (Ontario)) for
virus isolation and an aliquot of successfully cultivated virus,
generally after single passage, was submitted to the National
Microbiology Laboratory (Canada’s influenza virus reference
laboratory) for characterization by haemagglutination inhibition
(HI) assay [21]. Currently, an 8-fold or greater reduction in postinfection ferret HI-antibody titre raised to a given reference strain
and tested against a field isolate constitutes meaningful antigenic
distinction between reference and test viruses, although previously
a threshold of 4-fold or greater titre reduction had been applied
[21].
For H3N2 viruses, HI characterization was undertaken not only
relative to the A/Victoria/361/2011 virus passaged in MDCK
cells with whole HA identical to the WHO-recommended
MDCK-passaged vaccine prototype, but also relative to the eggpassaged version with whole HA identical to the IVR-165
reassortant vaccine strain. The former was conducted using turkey
erythrocytes and validated with guinea pig erythrocytes; the latter
was conducted with guinea pig erythrocytes directly [21].
A subset of sentinel H3N2 HA1 and A(H1N1)pdm09 HA1/
HA2 genes from viruses detected across the season and
contributing to VE analysis were sequenced for phylogenetic
and pair-wise AA identity comparison according to methods
described in Text S1. Virus was sequenced from culture isolates
(Ontario, per above) or original patient specimens (all provinces
including Ontario in the event virus could not be cultivated).
Genotypic findings were interpreted in relation to corresponding
phenotypic findings based on HI antigenic characterization. For
this analysis we referred to established antigenic site maps which
for H3 consist of 131 AA residues across antigenic sites A–E as
enumerated in Table S1 [18,19,22] and for H1 consist of 50 AA
residues across antigenic sites Sa, Sb, Ca1, Ca2, and Cb as also
enumerated in Table S1 [19,23].
Influenza B viruses were characterized at the lineage- and/or
strain-level by HI, phylogenetic analysis or an influenza B-lineagespecific one-step conventional RT-PCR assay [24]. Because
antigenic site maps for influenza B have not yet been established,
further gene sequencing and pair-wise identity analysis were not
undertaken for influenza B.
Influenza detection
The 2012–13 season showed an early November rise and
December/January peak in H3N2 activity followed by greater
A(H1N1)pdm09 and influenza B contributions thereafter
(Figure 2). Overall, influenza virus was detected in 652/1501
(43%) specimens tested (Table 2). For the 626/652 (95%)
influenza detections for which influenza A/subtype and influenza
B/lineage could be determined, 394 (63%) were H3N2, 79 (13%)
were A(H1N1)pdm09, and one was a dual H3N2 and
A(H1N1)pdm09 co-infection; 98 (16%) belonged to the B/
Yamagata vaccine-lineage and 54 (9%) belonged to the B/
Victoria non-vaccine-lineage (Table 2). The proportion immunized by age and influenza type, subtype and lineage is shown in
detail in Table S2.
VE estimates
Crude and adjusted-VE estimates are provided in Table 3.
Overall VE was 50% (95%CI: 33–63%) and against influenza A
was 45% (95%CI: 24–60%). Both estimates were driven by the
predominant H3N2 activity during the 2012–13 season for which
VE was 41% (95%CI: 17–59%). VE was 59% (95%CI: 16–80%)
against A(H1N1)pdm09. Against influenza B, VE was higher at
68% (95%CI: 44–82%): 67% (95%CI: 30–85%) for B/Yamagata
vaccine-lineage and 75% (95%CI: 29–91%) for B/Victoria nonvaccine lineage viruses.
VE estimates were generally increased with restriction to those
without comorbidity and among children, but reduced with
restriction to adults only, notably those 20–49 years of age
(Table 3). Repeat immunization had varying effects: those who
had received both 2012–13 and 2011–12 TIV had lower VE
estimates against H3N2 than those who received 2012–13 TIV
alone (Table S3). Conversely, those immunized both seasons
showed higher protection against both influenza B/lineages. In
each of these sub-analyses, however, confidence intervals were
VE analysis
A specimen collected between November 1, 2012 (week 44) and
April 30, 2013 (week 18) was considered a case if it tested positive
for influenza virus and a control if it tested negative for all
influenza types/subtypes. Patients for whom the timing of
vaccination was unknown or ,2 weeks before symptom onset,
or for whom comorbidity was unknown were excluded. We
estimated the odds ratio (OR) for medically-attended, laboratoryconfirmed influenza in vaccinated versus non-vaccinated participants by logistic regression with adjustment for clinically-relevant
confounders. Per previous VE analyses from this sentinel system,
covariates included age, comorbidity, province, week of specimen
collection and the interval between ILI onset and specimen
collection [1,12–19]. VE was calculated as [1-adjustedOR]6100.
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2012–13 Influenza Vaccine Effectiveness
Figure 1. Specimen exclusion for influenza vaccine effectiveness analysis, Canada, 2012–13 sentinel surveillance system. NOTE:
exclusions shown here in stepwise fashion to arrive at total case and control tally (i.e. those meeting multiple exclusion criteria are counted on the
basis of the first exclusion criterion met in the list shown). Missing collection dates were imputed as the laboratory accession date minus two days,
the average time period between collection date and laboratory accession date for records with valid data for both fields.
doi:10.1371/journal.pone.0092153.g001
site, include H156Q and G186V substitutions at antigenic site B,
and S219Y mutation at antigenic site D. Conversely, the HA1
gene of all 152 circulating viruses, like their 2011–12 vaccine and
circulating predecessors [19], shared AA identity with the MDCKcell-passaged prototype at these three positions (Table 4). In
association with the IVR-165 antigenic-site mutations, we
observed 16-fold reduction in HI antibody titre raised against
the egg-passaged version when tested against the MDCK-cellpassaged prototype. This is consistent with the 32-fold reduction
also reported by the WHO in its comparison between IVR-165
and the WHO-recommended prototype [10]. Also similar to the
WHO report [10], there was no reduction for antibody raised to
the MDCK-cell-passaged virus when tested in reverse against the
egg-passaged version in two-way HI comparison.
There were 132 H3N2 isolates successfully cultured for HI
characterization, with collection dates spanning November 20 to
April 10, including 61 (46%) with November–December, 68 (52%)
with January–February and 3 (2%) with March–April collection.
None of these isolates showed $8-fold reduction in antibody titre
relative to the MDCK-cell-passaged strain, indicating that
circulating viruses spanning the H3N2 season were antigenicallyequivalent to the WHO-recommended prototype (Table 2).
Conversely, all but one H3N2 isolate showed $8-fold reduction
relative to the egg-passaged strain, including viruses collected
from season start and with more than half of the circulating viruses
(72/130) showing 16-fold and one-quarter (34/130) showing
broad and overlapping. We particularly lacked statistical power
related to the A(H1N1)pdm09 component, although in separatelygrouped indicator analysis there was suggestion of comparable or
higher 2012–13 TIV protection in those with prior receipt of the
same unchanged A(H1N1)pdm09 vaccine antigen, including the
2009 monovalent formulation (Table S4).
Influenza genetic and antigenic characterization
To assess the impact of genotypic and phenotypic differences on
VE estimates, we compared antigenic site sequence analysis and
HI characterization of MDCK cell- and egg-passaged influenza A
vaccine and circulating viruses.
A(H3N2). Of 395 H3N2 infections diagnosed by PCR, 152
(38%) viruses contributed to genotypic analysis with specimen
collection dates spanning November 10 to April 10 including 56
(37%) with November–December, 92 (60%) with January–
February and 4 (3%) with March–April collection. The vast
majority of these viruses (143/152; 94%) belonged to the same
phylogenetic clade 3C as did both the MDCK-passaged, WHOrecommended A/Victoria/361/2011 prototype virus and the eggadapted IVR-165 reassortant strain actually used in vaccine
production (Figure S1).
However, more detailed sequence analysis revealed three
antigenic site AA differences between the IVR-165 and the
WHO-recommended A/Victoria/361/2011 prototype. These
three mutations in IVR-165, located close to the receptor binding
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2012–13 Influenza Vaccine Effectiveness
Table 1. Profile of participants included in primary influenza VE analysis, 2012–13, Canada.
Case (test-positive)
Characteristics
Age group (years)
Control (test-negative)
Total
N = 652; n (%)
N = 849; n (%)
N = 1501; n (%)
1–8
104 (16)
96 (11)
200 (13)
9–19
98 (15)
86 (10)
184 (12)
20–49
279 (43)
401 (47)
680 (45)
50–64
118 (18)
177 (21)
295 (20)
$65
53 (8)
89 (10)
142 (9)
Median age in years (range)
33 (1–92)
37 (1–95)
35 (1–95)
369 (57)
505 (59)
874 (58)
Female sex
Comorbiditya
112 (17)
187 (22)
299 (20)
Received 2012–13 TIVb
$2 weeks before symptom onset
95 (15)
224 (26)
319 (21)
Among:
those without comorbidity
55 (10)
138 (21)
193 (16)
those with comorbidity
40 (36)
86 (46)
126 (42)
1–8 years
5 (5)
18 (19)
23 (12)
9–19 years
0 (0)
10 (12)
10 (5)
20–49 years
36 (13)
73 (18)
109 (16)
50–64 years
26 (22)
57 (32)
83 (28)
$65 years
28 (53)
66 (74)
94 (66)
Yes
11 (39)
19 (29)
30 (32)
No
4 (14)
26 (39)
30 (32)
Unknown
13 (46)
21 (32)
34 (36)
c
2011–12 TIV
148/619 (24)
262/784 (33)
410/1403 (29)
2010–11 TIVd
151/596 (25)
267/752 (36)
418/1348 (31)
2009 A(H1N1)pdm09 vaccinee,f
240/556 (43)
331/709 (47)
571/1265 (45)
#4
522 (80)
623 (73)
1145 (76)
5–7
130 (20)
226 (27)
356 (24)
Median interval in days (range)
3 (0–7)
3 (0–7)
3 (0–7)
Among:
Adjuvanted vaccine ($65 years old)
Received prior influenza vaccine
Specimen collection
interval (days)
TIV = trivalent influenza vaccine; VE = vaccine effectiveness.
. Including any one or more of heart, pulmonary, renal, metabolic, blood, cancer, or conditions that compromise immunity or the management of respiratory
secretions, or morbid obesity [20].
b
. For the 2012–13 season, of 319 participants reporting vaccine receipt $2 weeks before symptom onset, 298 reported this was given through injection, 5 through
nasal spray (all children except one) with route of administration unspecified for 16.
c
. Children ,2 years of age in 2012–13 were excluded from 2011–12 vaccine uptake analysis as they may not have been vaccine-eligible during the fall 2011–12
immunization campaign on the basis of age ,6 months.
d
. Children ,3 years of age in 2012–13 were excluded from 2010–11 vaccine uptake analyses.
e
. In Canada, AS03-adjuvanted monovalent A(H1N1)pdm09 vaccine comprised .95% of doses distributed [17].
f
. Children ,4 years of age in 2012–13 were excluded from monovalent A(H1N1)pdm09 vaccine uptake analyses.
doi:10.1371/journal.pone.0092153.t001
a
and phenotypic (HI) characterization were undertaken there was a
similar distribution of up to 32-fold-reduction in HI titres relative
to the egg-passaged strain. This was true regardless of the nature
or number of additional AA mutations in circulating clade 3 or
clade 6 viruses beyond the three vaccine mutations (Table 5).
Taken together, these findings suggest that H3N2 viruses were
antigenically equivalent to the WHO-recommended prototype but
antigenically distinct from the IVR-165 vaccine component and
that vaccine mismatch was predominantly related to mutations in
the egg-adapted vaccine strain, rather than evolutionary drift in
circulating viruses.
A(H1N1)pdm09. Sequence analysis showed that the eggadapted X-179A (and X-181) vaccine reassortant strain bore no
antigenic-site AA mutations relative to the WHO-recommended
prototype, and of the 40 A(H1N1)pdm09 isolates spanning
November 19 to March 22 characterized by HI (85% collected
in January–February), all were antigenically-similar to the vaccine
32-fold titre reduction. This indicates that circulating viruses
spanning the season were antigenically distinct from IVR-165.
This antigenic separation between IVR-165 and circulating
viruses was primarily associated with mutations in the egg-adapted
vaccine. The most prevalent antigenic-site differences between
circulating viruses and IVR-165 are illustrated in Figure 3
[27,28], including the 3 differences from vaccine at positions 156,
186 and 219 resulting from IVR-165 mutation but not evident in
relation to the WHO-recommended prototype. The majority of
the circulating clade 3C viruses (134/144; 93%) showed a total of
5-7AA antigenic-site differences relative to IVR-165 (95–96%
vaccine identity) (Table 4). Fewer showed 4AA (4/144;3%) or
8AA (6/144;4%) total differences relative to IVR-165. Nine other
circulating H3N2 viruses belonged to clade 6 and showed
11–12AA antigenic-site differences from IVR-165 (91–92%
vaccine identity). However, among the 73 H3N2 viruses spanning
November 20 to April 10 for which both genotypic (sequencing)
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2012–13 Influenza Vaccine Effectiveness
Figure 2. Influenza specimens by week and subtype, 2012–13 sentinel surveillance period (N = 1682). NOTE: excludes specimens from
patients failing to meet the influenza-like illness case definition or unknown; specimens collected .7 days after influenza-like illness onset or interval
unknown; comorbidity unknown; age unknown or ,1 year and influenza test results unavailable or inconclusive on typing. Missing collection dates
were imputed as the laboratory accession date minus two days, the average time period between collection date and laboratory accession date for
records with valid data for both fields. One specimen diagnosed with both A/H3N2 and A(H1N1)pdm09 in week 2 is not presented in the graph.
Vaccine effectiveness analysis spans week 44 to week 18.
doi:10.1371/journal.pone.0092153.g002
strain (Table 2). Fifty-seven circulating A(H1N1)pdm09 viruses
spanning November 19 to March 8 (85% collected in January–
February) were also sequenced, of which 55 (96%) belonged to
clade 6 (Figure S2). In September 2013, the European Centre for
Disease Prevention and Control (ECDC) further divided clade 6
viruses into three genetic subgroups such that 1/55 (2%), 2/55
(4%) and 52/55 (95%) of our sentinel clade 6 A(H1N1)pdm09
viruses during 2012–13 belong to clade 6A, 6B and 6C,
respectively [29]. The majority of the sentinel clade 6 viruses
(47/55; 85%) showed 3AA antigenic-site substitutions relative to
X-179A/X-181 and 94% vaccine identity with fewer showing
2AA (7/55) or 4AA (1/55) substitutions and two clustered within
clade 7 with 3-4AA mutations (Table S5).
The genetic profile of circulating A(H1N1)pdm09 viruses differs
from 2011–12 when 90% of sequenced viruses clustered within
clade 7, bearing the same 2AA mutations shared by all subsequent
2012–13 clade 6/7 viruses (S185T/P and S203T) [19] but with
greater additional genetic diversity observed in 2012–13. Other
antigenic site mutations in 2012–13, located close to the receptorbinding site, include 21/57 (37%) viruses with R205K (seen in
clade 5 sequences in 2011–12), 17/57 (30%) with A141T, and
8/57 (14%) with A186T (Table S5, Figure S3) [30].
for which VE was first reported to be suboptimal (45%) in midseason publication [1] despite widespread laboratory reporting
that circulating viruses remained antigenically well conserved [2–
5,10]. In end-of-season analysis we corroborate mid-season
epidemiologic findings of low VE (41%) and reconcile these with
laboratory findings. Through detailed gene sequencing and HI
comparison we show that reduced vaccine protection during the
2012–13 season was related to mutations in the egg-adapted
H3N2 high growth reassortant strain used in vaccine production,
not antigenic drift in circulating viruses.
Vaccine match/mismatch to explain variable VE has historically focused on diversity and drift in circulating viruses and their
evolving antigenic distance from the corresponding vaccine
component. Here, we broaden that perspective to include the
potentially serious implications of even a few AA mutations
introduced through egg-adaptation of the WHO-recommended
cell-passaged prototype virus. The early provision of an eggadapted high growth reassortant version of the WHO-recommended prototype is a fundamental requirement of influenza
vaccine manufacturing, needed for further high-yield growth in
embryonated hens’ eggs as part of annual mass production [31].
However, in a variety of animal models, mammalian cell-derived
H1 and H3 viruses have been shown to induce more cross-reactive
antibody response and better protection than corresponding eggadapted variants bearing as few as 1–2AA mutations [32–35].
Such changes with egg passage, particularly if located near the HA
receptor-binding site, have been shown to dramatically alter
vaccine antigenicity, immunogenicity and efficacy [32–35].
Located closest to the receptor-binding site, mutations at antigenic
sites A, B and D of the H3 globular head are typically considered
most consequential [36] and the immuno-dominance of antigenic
Discussion
The sentinel surveillance system in Canada directly links
genotypic and phenotypic characterization of circulating influenza
viruses to epidemiologic measurement of VE in order to better
understand vaccine protection in the context of vaccine-virus
relatedness. For the 2012–13 season, we used this platform to
investigate protection provided by the H3N2 vaccine component,
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2012–13 Influenza Vaccine Effectiveness
Table 2. Laboratory profile, 2012–13 sentinel season.
Alberta
British
Columbia
Manitoba
Ontario
Québec
Total
Specimens included
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
Influenza tested (N)
450
319
114
337
281
1501
Influenza negative
Influenza positive
Influenza A positive
Influenza B positive
267 (59)
185 (58)
77 (68)
193 (57)
127 (45)
849 (57)
All influenza positive
183 (41)
134 (42)
37 (33)
144 (43)
154 (55)
652 (43)
A positive
122 (67)
102 (76)
28 (76)
114 (79)
119 (77)
485 (74)
B positive
61 (33)
32 (24)
9 (24)
30 (21)
35 (23)
167 (26)
A/H3N2
95 (78)
84 (82)
24 (86)
87 (76)
104 (87)
394 (81)
A(H1N1)pdm09
21 (17)
17 (17)
2 (7)
25 (22)
14 (12)
79 (16)
A/H3N2 & A(H1N1)pdm09
0
0
1 (4)
0
0
1 (1)
Subtype unknown
6 (5)
1 (1)
1 (4)
2 (2)
1 (1)
11 (2)
B/Yamagata (vaccine)
27 (44)
15 (47)
1 (11)
26 (87)
29 (83)
98 (59)
B/Victoria (non-vaccine)
27 (44)
14 (44)
7 (78)
3 (10)
3 (9)
54 (32)
Lineage unknown
7(12)
3(9)
1(11)
1(3)
3(9)
15(9)
HI Characterization (post-infection ferret anti-sera raised against reference virus tested against field isolate)
A/Victoria/361/2011 (MDCK)a
2
40
0
17
73
132
,4-fold reduced titre
2 (100)
38 (95)
0
11 (65)
37 (51)
88 (67)
$4-fold reduced titre
0
2 (5)
0
6 (35)
36 (49)
44 (33)
$8-fold reduced titre
0
0
0
0
0
0
A/Victoria/361/2011 (egg)b
0
0
0
0
1
1
,4-fold reduced titre
0
0
0
0
0
0
$4-fold reduced titre
2 (100)
40 (100)
0
16 (100)
72 (100)
130
$8-fold reduced titre
2 (100)
40 (100)
0
16 (100)
71 (99)
129 (99)c
A(H1N1)pdm09
Reference Virus
A/California/7/2009-like
1
8
0
24
7
40
Influenza B
Reference Virus
B/Wisconsin/01/2010
(Yamagata)d
22 (48)
11 (52)
0
15 (83)
25 (89)
73 (65)
B/Brisbane/60/2008 (Victoria)e
24 (52)
10 (48)
0
3 (17)
3 (11)
40 (35)
H3N2 Reference Virus
TIV: trivalent influenza vaccine; HI: haemagglutination inhibition assay.
. H3N2 prototype reference strain recommended as 2012–13 TIV component by the World Health Organization (WHO), as passaged in Madin Darby canine kidney cells;
assessed using turkey erythrocytes, validated with guinea pig erythrocytes.
b
. 2012–13 H3N2 vaccine strain as passaged in eggs and with HA1 sequence identical to the A/Victoria/361/2011 IVR-165 egg-adapted high growth reassortant vaccine
strain; assessed based on guinea pig erythrocytes.
c
. Nineteen of the 129 viruses (19%) manifesting $8-fold reduction had been collected from vaccinated participants, comparable to the proportion immunized among
H3 detections overall (17%) and among whom 12/19 (63%) showed 16-fold and 4/19 (21%) showed 32-fold reduction.
d
. 2012–13 TIV component.
e
. 2011–12 TIV component.
doi:10.1371/journal.pone.0092153.t002
a
193) have been highlighted as responsible for all major H3N2
antigenic cluster transitions since 1968 [40]. Of these, only
position 156 distinguishes circulating viruses in 2012–13 from
IVR-165 and not from the WHO-recommended cell-passaged
prototype. Although our circulating viruses also manifest substitution at position 145 in relation to both IVR-165 and the WHO
prototype (Table 4), this difference did not exacerbate foldreduction in HI titres in relation to the former, and did not alter
antigenic equivalence in relation to the latter.
Divergence at position 156 due to vaccine mutation may have
therefore been particularly influential in reducing antibody
recognition and neutralization of circulating viruses, compromising VE. Of note, the A/Texas/50/2012 egg-adapted high growth
reassortant strain (X-223) selected as replacement for the 2013–14
TIV also manifests substitutions at positions 186 and 219 (Table 4)
but no longer at position 156. With that 156 homology, X-223
shows antigenic equivalence (#4-fold reduction in HI titres)
site B is particularly emphasized among more recent H3N2 strains
[37].
In that regard, we highlight mutations present in the 2012–13
egg-adapted high growth reassortant IVR-165 vaccine strain
relative to the WHO-recommended H3N2 prototype at positions
156 and 186 of site B, and at position 219 of site D. These
mutations were associated with altered vaccine antigenicity and
low VE even while antigenic integrity of circulating viruses was
maintained. Site B positions 156 and 186 are well-known eggadaptation sites [38,39] but QH (i.e. glutamine-histidine) variation
at position 156 has additionally been highlighted as one of two HA
residues (in addition to position 155) responsible for the significant
A/Fujian/411/02 (H3N2) antigenic drift and the suboptimal VE
reported during that dramatic 2003–04 influenza epidemic [38].
In more recent publication, substitutions at just seven of the 131
H3N2 A–E antigenic site residues, located exclusively in antigenic
sites A (position 145) and B (positions 155, 156, 158, 159, 189 and
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Table 3. Primary and restricted analysis - influenza vaccine effectiveness based on sentinel system in Canada 2012–13 season.
Vaccine Effectiveness % (95% Confidence Interval)
Covariates and adjustment
Any Influenza
Influenza A and Subtype specific
Influenza B and Lineage specific
Any
Influenza A
A/H3N2
A/H1N1pdm09
Any
Influenza B
B/Yamagata (vaccine)
B/Victoria
(non-vaccine)
Primary analysis N total
1501
1334
1244
929
1016
947
903
[n Cases; n vaccinated]
[652; 95]
[485; 78]
[395; 66]
[80; 10]
[167; 17]
[98; 9]
[54; 5]
(n Controls; n vaccinated)
(849; 224)
(849; 224)
(849; 224)
(849; 224)
(849; 224)
(849; 224)
(849; 224)
Unadjusted
52 (38–64)
47 (29–60)
44 (24–59)
60 (21–80)
68 (47–81)
72 (43–86)
72 (28–89)
Age (1–8, 9–19, 20–49, 50–64,
$65 years)
51 (35–63)
46 (26–60)
44 (22–60)
56 (10–79)
68 (44–82)
67 (31–85)
76 (32–92)
Comorbidity (yes/no)
51 (35–63)
45 (27–59)
43 (22–58)
59 (19–80)
66 (43–80)
71 (40–86)
68 (18–88)
Province (BC, AB, MB, ON, QC)
52 (37–63)
46 (28–60)
43 (23–58)
59 (20–80)
69 (47–81)
72 (43–86)
72 (28–89)
Specimen collection interval
(#4 d/5–7 d)
52 (37–63)
46 (28–60)
42 (21–58)
62 (25–81)
68 (46–81)
72 (43–86)
71 (25–89)
Week of illness onset
52 (37–63)
45 (27–59)
41 (20–57)
62 (24–81)
69 (48–82)
73 (45–87)
71 (27–89)
Age, comorbidity, province,
interval, week
50 (33–63)
45 (24–60)
41 (17–59)
59 (16–80)
68 (44–82)
67 (30–85)
75 (29–91)
8
Restricted to participants with no comorbidity
N total; n Cases; n Controls
1202; 540; 662
1059; 397; 662
984; 322; 662
728; 66; 662
805; 143; 662
745; 83; 662
710; 48; 662
Adjusteda
60 (43–72)
59 (38–72)
53 (28–69)
80 (40–93)
70 (40–85)
69 (25–88)
68 (24–90)
384; 202; 182
315; 133; 182
301; 119; 182
193; 11; 182
251; 69; 182
228; 46; 182
203; 21; 182
87 (65–95)
84 (53–95)
87 (55–96)
NE
91 (35–99)
88 (7–98)
NE
Restricted to participants age 1–19 years old
N total; n Cases; n Controls
Adjusted
b
Restricted to participants age 20–49 years old
680; 279; 401
622; 221; 401
567; 166; 401
451; 50; 401
459; 58; 401
429; 28; 401
424; 23; 401
Adjustedc
31 (28–56)
32 (210–58)
17 (240–51)
56 (217–84)
32 (260–71)
10 (2181–71)
54 (2103–90)
Restricted to participants age $50 years old
N total; n Cases; n Controls
437; 171; 266
397; 131; 266
376; 110; 266
285; 19; 266
306; 40; 266
290; 24; 266
276; 10; 266
Adjustedd
47 (17–66)
35 (26–60)
32 (215–59)
52 (251–85)
65 (22–84)
73 (11–92)
79 (4–96)
BC = British Columbia, AB = Alberta, MB = Manitoba, ON = Ontario, QC = Québec; d = days; NE = not estimable owing to sparse data.
. Adjusted for age (1–8, 9–19, 20–49, $50 years), province, interval, week.
. Adjusted for age (1–8, 9–19 years), comorbidity, province, interval, week; except B/Yamagata not adjusted for province.
c
. Adjusted for comorbidity, province, interval, week; except B/Victoria not adjusted for province.
d
. Adjusted for age (50–64, $65 years), province, interval, week; except A(H1N1)pdm09, influenza B, B/Victoria, B/Yamagata not adjusted for province.
doi:10.1371/journal.pone.0092153.t003
a
b
2012–13 Influenza Vaccine Effectiveness
March 2014 | Volume 9 | Issue 3 | e92153
N total; n Cases; n Controls
PLOS ONE | www.plosone.org
Table 4. Haemagglutinin antigenic site differences in circulating H3N2 viruses relative to the 2012–13 egg-adapted A/Victoria/361/2011 IVR-165a high growth reassortant vaccine
strain.
H3N2 Hemagglutinin
Vaccine Reference Virus = Victoria
361 IVR-165
Antigenic Site
C
E
D
A
B
A
B
D
E
C
# of AA
Clade differencesb
% AA
identityb
HA1 Position
45 48 53 54 62 67 88 94 103 121 124 128 140 142 145 156 157 186 192 193 198 219 226 230 262 278 280 304 312
A/Victoria/210/2009 (X-187)c
N
T
D
S
K
I
V
Y
P
N
S
T
I
R
N
H
L
V
I
F
S
S
I
I
S
N
E
A
N
1
11
91.6%
A/Victoria/361/2011 (MDCK)
97.7%
N
I
D
S
E
I
V
Y
P
N
S
T
I
R
N
H
L
G
I
F
S
S
I
I
S
N
E
A
S
3C
3
A/Victoria/361/2011 (IVR-165)a N
I
D
S
E
I
V
Y
P
N
S
T
I
R
N
Q
L
V
I
F
S
Y
I
I
S
N
E
A
S
3C
-
-
A/Texas/50/2012 (MDCK)d
N
I
D
S
E
I
V
Y
P
N
S
N
I
R
N
H
L
G
I
F
P
S
I
I
S
K
E
A
S
3C
6
95.4%
N
I
D
S
E
I
V
Y
P
N
S
N
I
R
N
H
L
V
I
F
P
F
N
I
S
K
E
A
S
3C
6
95.4%
A/Texas/50/2012 (X-223)d
British Columbia
N
9
10
A
G
S
H
G
S
K
3C
7
94.7%
12
A
G
S
H
G
S
K
3C
7
94.7%
A/British Columbia/023/2012
1
A/British Columbia/002/2013
6
S
H
A/British Columbia/022/2013
1
A/British Columbia/023/2013
1
A/British Columbia/025/2013
1
Alberta
N
A/Alberta/053/2012
2
A/Alberta/054/2012
1
A/Alberta/056/2012
21
A/Alberta/059/2012
13
A/Alberta/060/2012
4
A/Alberta/02/2013
1
A/Alberta/03/2013
2
A/Alberta/06/2013
1
A/Alberta/24/2013
1
Manitoba
N
A/Manitoba/001/2012
2
A/Manitoba/003/2012
5
A/Manitoba/004/2012
3
A/Manitoba/01/2013
1
Ontario
N
A/Ontario/030/2012
9
H
A
T
N
K
3C
5
96.2%
K
3C
5
96.2%
S
H
G
S
K
3C
8
93.9%
S
H
G
S
K
3C
6
95.4%
S
H
G
S
K
3C
6
95.4%
H
G
3C
4
96.9%
H
G
6
12
90.8%
S
H
G
S
K
3C
6
95.4%
S
H
G
S
K
3C
5
96.2%
S
H
G
S
K
3C
6
95.4%
V
G
S
V
G
V
V
A
S
S
S
H
A
G
G
G
Q
S
S
G
S
A
S
S
H
G
S
S
H
G
S
S
H
G
S
H
G
S
K
K
V
A
N
D
N
K
3C
6
95.4%
K
3C
7
94.7%
S
K
3C
7
94.7%
S
K
3C
7
94.7%
S
H
G
S
K
3C
6
95.4%
S
H
G
S
K
3C
5
96.2%
S
H
G
S
K
3C
7
94.7%
S
H
G
S
3C
4
96.9%
S
H
G
S
3C
5
96.2%
K
2012–13 Influenza Vaccine Effectiveness
March 2014 | Volume 9 | Issue 3 | e92153
A/British Columbia/020/2012
A/British Columbia/021/2012
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Table 4. Cont.
H3N2 Hemagglutinin
Vaccine Reference Virus = Victoria
361 IVR-165
Antigenic Site
C
HA1 Position
45 48 53 54 62 67 88 94 103 121 124 128 140 142 145 156 157 186 192 193 198 219 226 230 262 278 280 304 312
A/Ontario/031/2012
2
A/Ontario/001/2013
1
A/Ontario/004/2013
1
10
A/Ontario/005/2013
8
A/Ontario/015/2013
1
A/Ontario/018/2013
1
A/Ontario/021/2013
1
A/Ontario/038/2013
1
Quebec
N
A/Quebec/011/2012
22
A/Quebec/012/2012
1
A/Quebec/016/2012
4
A/Quebec/019/2012
3
A/Quebec/020/2012
2
A/Quebec/021/2012
2
1
2
A/Quebec/14/2013
1
A/Quebec/26/2013
1
D
A
R
B
A
A
B
G
S
S
T
N
G
T
N
H
S
T
N
H
T
N
% AA
identityb
93.9%
H
G
S
K
3C
8
H
G
S
K
3C
6
95.4%
H
G
6
11
91.6%
A
S
V
A
N
S
K
3C
7
94.7%
A
S
K
3C
6
95.4%
S
H
G
H
G
K
S
H
G
S
K
3C
6
95.4%
H
G
S
K
3C
4
96.9%
S
G
S
H
G
S
K
3C
7
94.7%
A
G
94.7%
S
H
G
S
K
3C
7
S
H
G
S
K
3C
6
95.4%
H
G
6
11
91.6%
3C
5
96.2%
6
11
91.6%
3C
5
96.2%
A
G
S
V
S
A
S
A
N
A
N
K
G
S
H
G
H
G
G
S
H
G
S
H
G
S
K
3C
6
95.4%
G
S
H
G
S
K
3C
8
93.9%
M
A
S
T
H
Q
G
# of AA
Clade differencesb
S
H
S
C
S
I
S
E
S
H
A
D
V
S
T
V
S
S
K
V
A
K
N
6
11
91.6%
3C
8
93.9%
N = number of sentinel viruses with that sequence. Bold font signifies amino acid (AA) substitutions compared with IVR-165. Clade designation, number of antigenic site differences and percent antigenic site pairwise identity are also
displayed. Only the 31/131 antigenic site positions showing differences between circulating H3N2 viruses and IVR-165 are displayed. AA sequences at those positions for other recent vaccine viruses are also displayed.
a
. IVR-165 is the egg-adapted high growth reassortant strain substituted by manufacturers for the MDCK-passaged A/Victoria/361/2011 (H3N2) prototype virus recommended as 2012–13 vaccine component by the World Health
Organization (WHO) designated here as A/Victoria/361/2011 (MDCK).
b
. Number of antigenic site AA differences and percent antigenic site identity relative to IVR-165. Percent identity derived as per Text S1.
c
. A/Victoria/210/2009 (X-187) is the egg-adapted high growth reassortant strain used by manufacturers for the 2011–12 influenza vaccine for the northern hemisphere., shown for historic comparison.
d
. A/Texas/50/2012 (MDCK) and (X-223) are the WHO-recommended prototype and egg-adapted high growth reassortant strains, respectively, for the 2013–14 influenza vaccine for the northern hemisphere, shown for added
comparison.
doi:10.1371/journal.pone.0092153.t004
2012–13 Influenza Vaccine Effectiveness
March 2014 | Volume 9 | Issue 3 | e92153
A/Quebec/034/2012
A/Quebec/038/2012
E
2012–13 Influenza Vaccine Effectiveness
Figure 3. Three-dimensional model of antigenic-site differences between circulating H3N2 viruses and the 2012–13 egg-adapted A/
Victoria/361/2011 IVR-165 high growth reassortant vaccine strain. One HA1 monomer is shown with five previously defined antigenic site
residues of A–E colored in light green, dark green, light blue, dark blue and purple, respectively, mapped onto a related crystal structure (A/X31(H3N2), PDB, 1HGG) [27] using PyMOL [28]. The most prevalent antigenic site amino acid differences between circulating clade 3C viruses in
Canada relative to the egg-adapted A/Victoria/361/2011 IVR-165 vaccine reassortant strain are shown in red and labelled with coloured font
representing their antigenic sites, viewed from the front (A) or side (B). Three amino acid differences (Q156H, V186G and Y219S) are owing to
mutation in the egg-adapted IVR-165 vaccine strain rather than circulating viruses which instead share identity with the MDCK-passaged WHO
reference prototype at these positions. RBS indicates approximate location of the receptor-binding site.
doi:10.1371/journal.pone.0092153.g003
relative to the MDCK cell-passaged A/Victoria/361/2011 strain
that is once again the WHO-recommended prototype for the
2013–14 vaccine [10]. It is concerning, however, that 74% (90/
122) of our sentinel viruses collected during the 2012–13 season
still showed $8-fold reduction in HI titres when further tested with
anti-sera raised against the egg-passaged A/Texas/50/2012 strain
and 24% (29/122) showed $16-fold reduction. X-223 manifests
additional antigenic site B (T128N, S198P) and D (I226N)
mutations relative to A/Victoria/361/2011 and IVR-165, different also from our circulating viruses. Ongoing monitoring of
H3N2 vaccine-virus relatedness and impact on VE thus remain
critical.
These findings related to mutation in egg-adapted vaccine
strains highlight a need for in-depth monitoring not only of
circulating viruses but also of annual vaccine constituents. In
reporting vaccine match/mismatch, both real time [2–5] and in
retrospective reviews [41], the comparator vaccine referent
(whether the original MDCK or egg-passaged WHO prototype,
egg-adapted high growth reassortant strain or further eggpropagated virus) should be specified. This would enable more
accurate understanding of the correlation between antigenic
match and VE. Until now, commentaries on VE as it relates to
vaccine match have focused on the similarity between circulating
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virus and the WHO recommended reference—an approach that
our study shows can lead to incorrect conclusions about similarity
to the actual vaccine component used and the anticipated vaccine
protection on that basis. While evolutionary drift in circulating
viruses cannot be regulated, mutations that are introduced as part
of egg-based vaccine production may be amenable to improvements. To determine the antigenic relationship between two
viruses proposed as equivalent vaccine candidates, ferret anti-sera
to both viruses (e.g. the egg- and MDCK-passaged) must be used
in a ‘‘two-way’’ HI test [21]. In the current study, and in follow-up
report by the WHO [10], two-way HI testing revealed $8-fold
reduction in antibody titre raised to the egg-passaged strain when
tested against the MDCK-passaged version, but this titre reduction
was not observed when tested in reverse (anti-sera raised to the
MDCK-passaged strain tested against the egg-passaged virus).
One-way HI testing consisting only of the latter more crossreactive direction does not show the antigenic difference between
IVR-165 and the recommended cell-passaged A/Victoria/361/
2011 prototype [11]. Routine display of two-way HI testing for
candidate vaccine viruses could reveal this issue in advance of
vaccine production and use, and enable public health programs to
more broadly respond to its potential implications.
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2012–13 Influenza Vaccine Effectiveness
Table 5. Distribution of fold-reduction in haemagglutination inhibition (HI) titres relative to the 2012–13 egg-passaged H3N2
strain by nature and location of additional amino acid (AA) mutations present in HA1 antigenic sites of circulating viruses.
Specific HA1 antigenic site AA mutations in circulating viruses by fold-reduction in HI titre
relative to the egg-passaged H3N2 vaccine straina
Number of additional antigenic
site AA mutations in circulating
virusesb (N = number of viruses)
Clade
4-fold
n/N (%)
n/N (%)
n/N (%)
n/N (%)
1 (N = 1)
3C
—
—
—
1/1 (100%)
2 (N = 16)
3C
—
3/16 (19%)
8/16 (50%)
5/16 (31%)
N278K +
N278K +
N278K +
N145S (64) or
N145S (65)
8-fold
16-fold
32-fold
N278K [C]
N145S [A] (63)
L157S [B] (64)
3 (N = 4)
3C
—
—
1/4 (25%)
3/4 (75%)
N278K +
N278K +
N145S +
N145S +
V88I [E] (61)
S54G [C] (61) or
S198A [B] (61) or
I140M [A] (61)
4 (N = 44)
3C
1/44 (2%)
10/44 (23%)
25/44 (57%)
8/44 (18%)
N278K +
N278K +
N278K +
N278K +
N145S +
N145S +
N145S +
N145S +
R142G [A] +
R142G +
R142G +
R142G +
T128A [B]
T128A (69) or
T128A (625)
T128A (68)
—
—
1/2 (50%)
1/2 (50%)
T128S (61)
5 (N = 2)
8 (N = 6)
3C
6
—
—
N278K +
N278K +
N145S +
N145S +
R142G +
R142G +
T128A +
T128A +
E62G [E] (61)
I192V [B] (61)
3/6 (50%)
3/6 (50%)
N45S [C] +
N45S +
I48T [C] +
I48T +
D53N [C] +
D53N +
I230V [D] +
I230V +
E280A [C] +
E280A +
S312N [C] +
S312N +
Y94H/Q [E] +
Y94H/Q +
S198A/T [B] (63)
S198A/T (63)
HA1 = haemagglutinin 1 protein.
Mutations are highlighted in bold in the first row that they are represented in the table.
HA1 antigenic site positions [A–E] affected are annotated in bold the first time they appear.
‘‘(x n)’’ following a specified amino acid residue indicates the number of viruses with that specific mutation.
a
. The 2012–13 egg-passaged H3N2 strain used in haemagglutination inhibition (HI) assay was identical in its HA1 to the egg-adapted A/Victoria/361/2011-IVR-165 high
growth reassortant vaccine strain.
b
. In addition to the 3 AA differences (at positions 156, 186, and 219) present in the egg-passaged H3N2 strain used in the HI assay and the egg-adapted A/Victoria/361/
2011 IVR-165 high growth reassortant vaccine strain.
doi:10.1371/journal.pone.0092153.t005
Specific virus-host interactions are also relevant to consider in
interpreting VE findings. In sub-analyses, VE was higher for all
TIV components in young participants ,20 years of age and those
without comorbidity, but for H3N2 was further reduced in adults
and those with history of prior immunization. Random variation
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associated with small sample size in subgroup analysis has to be the
first consideration in explaining these differences. Beyond that,
hypotheses to explain variability of repeat vaccine effects include
varying positive or negative interference from pre-existing
antibody determined by antigenic distance across successive
12
March 2014 | Volume 9 | Issue 3 | e92153
2012–13 Influenza Vaccine Effectiveness
vaccine and circulating variants as well as differential neutralization efficiency of affected HA epitopes [36,42]. Ongoing
monitoring of genetic variability across vaccines and circulating
viruses may improve resolution and refine our measure of
antigenic distance relevant to the effects of repeat immunization.
The previous season’s 2011–12 TIV included the antigenicallydistinct H3N2 predecessor strain A/Victoria/210/2009-X-187
bearing 11AA differences from IVR-165 and 91.6% cross-vaccine
identity (Table 4). We lacked statistical power to explore the
influence of prior immunization stratified by age or comorbidity
but among immunized controls, a comparable proportion ,20
years versus 20–49 years of age were immunized the prior year
(15/18, 83% versus 55/68, 81%; p = 0.19), greater among those
with than without comorbidity (79/82, 96% versus 101/124, 82%;
p,0.01). However, single cross-season differences in prior
immunization do not necessarily reflect the cumulative lifetime
effects of vaccine- or virus-induced antibody that may also be
influential. Such immunologic interactions are important to
explore but most studies, including our own, lack the required
power to assess their intricate effects.
Our end-of-season analyses provide other noteworthy insights.
Relative to the WHO-recommended prototype, there were no
antigenic-site mutations in the 2012–13 egg-adapted
A(H1N1)pdm09 X-179A (or X-181) vaccine strain. Circulating
viruses were shown by HI to remain antigenically similar to A/
California/07/2009, retained as vaccine antigen since 2009.
Nevertheless, our point estimate of VE in 2012–13 (59%;
95%CI: 16–80%) was reduced compared to the prior 2011–12
season (80%; 95%CI: 54–92%) [19]. Confidence intervals around
each of these estimates are broad and overlapping such that
conclusions regarding VE trends across seasons cannot be drawn.
However, the genetic profile of circulating A(H1N1)pdm09 viruses
in 2012–13 was more diverse than 2011–12, particularly in
relation to the receptor binding site. Ongoing monitoring of
differences in the contributing mix of genetic variants across
seasons and their correlation with variation in VE may be relevant
given recent resurgence of A(H1N1)pdm09 activity [43] and
retention of the same vaccine antigen for the northern hemisphere’s 2013–14 TIV. After including the same B/Victorialineage as TIV component across three consecutive seasons (2009–
10 to 2011–12), the WHO recommended a lineage-level switch to
B/Yamagata-containing vaccine for the 2012–13 TIV. We found
comparable VE estimates of about 70% for co-circulating
Yamagata- and Victoria-lineages this season. Immunologic
recognition across influenza B/lineages might be anticipated given
the greater AA similarity across the HA1 of influenza B/lineages
(,90% pairwise identity) than across influenza A H1/H3 subtypes
(,35% pairwise identity) [44]. We have previously demonstrated
cross-lineage immunologic interactions and differential vaccine
effects based on prior original priming and subsequent boost
exposure histories [44–46]. Population heterogeneity in B/lineage
exposures with differential recall of immunologic memory (i.e.
complex cohort effects) may be evident in cross-lineage protection
with varying age-related and prior immunization effects (Table
S3). Recent meta-analysis has summarized cross-lineage TIV
effectiveness from eight randomized controlled trials, mostly
among adults, at 52% (95%CI: 19–72%) [47]. Precise quantification and better understanding of the variability in cross-lineage
VE for influenza B will be crucial in assessing the incremental costbenefit of proposed quadrivalent vaccine formulations to replace
TIV.
There are limitations to this study. We routinely assess vaccinerelatedness through gene sequencing and HI characterization of
contributing viruses from across the season, but this represents
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only a proportion of all influenza virus detections. Systematic
differences in viruses available for characterization or sequencing
cannot be ruled out—an issue for all laboratory-based surveillance.
We did not directly access IVR-165 but instead, MDCK- and eggpassaged viruses used in HI assays were derived from reference
strains provided by the United States Centers for Disease Control
and Prevention, confirmed through sequence analysis to be
identical in their HA to the A/Victoria/361/2011 WHO
prototype and to the IVR-165 reassortant strains, respectively.
Two-way HI comparison of these viruses was consistent with
WHO report [10]. Working-age adults and repeat vaccine
recipients typically comprise the majority of our sample; virologic
and VE findings may not be generalizable to other populations. In
Canada, universal health care coverage addresses barriers to
access that may exist in other countries. We include only
participants meeting a specified ILI definition presenting within
7 days of ILI onset helping to standardize for health-care seeking
behaviour and illness severity. However, patient and clinician
discretion is still incorporated into the decision to test. Because no
national immunization registry documenting influenza vaccine
receipt exists in Canada, self-report of vaccine status cannot be
further validated but has been shown elsewhere to be reliable [48]
and was comparable here to separate survey estimates of coverage
for seasonal TIV (,30%) [25] and 2009 monovalent pandemic
H1N1 (,40%) [49] vaccine. We do not collect information on
manufacturer’s brand of vaccine administered, but most of the
seasonal vaccine publicly funded in Canada is non-adjuvanted
inactivated split virion product; other formulations are available
such as live attenuated vaccine preferentially recommended for
children or adjuvanted subunit vaccine approved for the elderly
[20], but as shown in Table 1, these products contributed little to
our overall or age-stratified 2012–13 VE analyses. Although we
conducted subset analyses of VE, the reduced sample size and
wide confidence intervals in sub-analyses preclude definitive
conclusions. Validity of VE estimates derived by the test-negative
approach has been demonstrated previously through modelling
[50] and more recently empirically through direct comparison to
gold-standard per-protocol analysis of the same randomizedcontrolled trial datasets [51]. Our participant profiles are
comparable to previous estimates from the sentinel system and
community surveys in Canada. Although we observed no obvious
flags for concern, as with any observational design, we cannot rule
out residual bias and confounding.
In summary, our findings underscore the need to monitor
vaccine viruses as well as circulating strains to explain vaccine
performance. Evolutionary drift in circulating viruses cannot be
regulated, but virus changes introduced as part of egg-based
vaccine production may be amenable to improvements. In that
regard a better understanding of specific mutations related to eggadaptation and most influential upon vaccine protection is needed.
We highlight the immuno-epidemiologic complexity that may
further influence VE, including agent-host interactions and prior
antigenic exposures. This complexity is daunting to consider but
critical to confront in improving influenza prevention and control.
Finally, we show that sentinel surveillance structures can efficiently
and reliably link detailed virologic and epidemiologic observations
at the molecular, individual and population levels in support of
programmatic and scientific insights and should be considered a
core requirement for ongoing influenza vaccine monitoring and
evaluation.
13
March 2014 | Volume 9 | Issue 3 | e92153
2012–13 Influenza Vaccine Effectiveness
Prior 2011–12 trivalent influenza vaccine (TIV)
effects on current 2012–13 TIV effectiveness.
(PDF)
Table S3
Supporting Information
Figure S1 Phylogenetic tree of influenza A/H3N2 viruses, sentinel system 2012–13. A maximum-likelihood phylogeny of the 152 sentinel viruses in the context of globally isolated
2012–2013 H3N2 viruses and recent vaccine components (n = 93)
based on nucleotide alignment of the haemagglutinin HA1
domain is shown. Vaccine components and previously reported
clades are labelled; sentinel viruses are coloured by province of
origin.
(PDF)
Prior 2011–12 trivalent influenza vaccine (TIV)
and/or 2009 monovalent pandemic vaccine effects on
2012–13 TIV effectiveness vs. A(H1N1)pdm09.
(PDF)
Table S4
Table S5 Haemagglutinin antigenic site mutations
in circulating A(H1N1)pdm09 viruses relative to the
2012–13 egg-adapted A/California/07/2009 X-179A high
growth reassortant vaccine strain.
(PDF)
Figure S2 Phylogenetic tree of influenza A(H1N1)pdm09
viruses, sentinel system 2012–2013. A maximum-likelihood
phylogeny of the 57 sentinel viruses in the context of globally
isolated 2012–2013 A(H1N1)pdm09 viruses and recent vaccine
components (n = 77) based on nucleotide alignment of the
haemagglutinin HA1/HA2 domains is shown. Vaccine components and previously reported clades are labelled; sentinel viruses
are coloured by province of origin. In September 2013, the
European Centre for Disease Prevention and Control (ECDC)
further divided clade 6 viruses into three genetic subgroups such
that 1/55 (2%), 2/55 (4%) and 52/55 (95%) of the sentinel clade 6
A(H1N1)pdm09 viruses displayed belong to clade 6A, 6B and 6C.
Subclade details are displayed in Table S5 [29].
(PDF)
Text S1 Methods for haemagglutinin sequencing, phylogenetic and percent identity analysis.
(PDF)
Acknowledgments
Authors recognize the invaluable contribution of sentinel sites and the
coordination and technical support provided by epidemiologic and
laboratory staff in all participating provinces. We wish especially to
acknowledge the provincial coordination provided by Quynh Le Ba and
Elaine Douglas for TARRANT in Alberta; Hazel Rona of the Winnipeg
Regional Health Authority, Manitoba; Romy Olsha and Elizabeth
Balogun for Public Health Ontario; and Monique Douville-Fradet, Sophie
Auger and Rachid Amini for the Institut national de santé publique du
Québec. We also recognize Kanti Pabbaraju, Sallene Wong and Danielle
Zarra of the Alberta Provincial Laboratory; Roy Cole of the National
Microbiology Laboratory and Cadham Provincial Laboratory, and Kerry
Dust of the Cadham Provincial Laboratory in Manitoba; Paul Rosenfeld
and Aimin Li of Public Health Ontario; and Joel Ménard and Lyne
Désautels of the Québec Provincial Laboratory for their contributions to
virus detection and sequencing. We thank Catharine Chambers of the BC
Centre for Disease Control for supporting literature review and summary.
Finally, we gratefully acknowledge the authors, originating and submitting
laboratories of the sequences obtained from GISAID’s EpiFlu Database
used in the phylogenetic analysis.
GenBank Accession Numbers
KC526204-KC526214 (excepting 208, 209 and 213); KC535019KC35064 (excepting 026, 030, 045, 047, 050, 058); KC539119KC539136 (excepting 121); KF761446-KF761513; KF850641KF850683; KF886348-KF886381.
Figure S3 Three-dimensional model of antigenic-site
mutations in circulating A(H1N1)pdm09 viruses relative
to the 2012–13 egg-adapted A/California/04/2009 X179A high growth reassortant vaccine strain. Threedimensional structures of the trimeric haemagglutinin (HA)
protein were constructed using the crystal structure of the A/
California/04/2009(H1N1) HA (PDB, 3LZG) [30] Amino acid
residues of the Sa, Sb, Ca1, Ca2 and Cb antigenic regions on the
molecular surface (A: front view; B: top view) are colour-coded
purple, green, yellow, pink and blue respectively and amino acid
substitutions in circulating viruses relative to X-179A are labelled
with coloured text representing their antigenic site positions. The
three most prevalent mutations found in this study are coloured
red (R205K site Ca1, A141T site Ca2, and A186T site Sb). Clade
characteristic mutations S185T (representative for S185T/P) in
antigenic site Sb and S203T (not visible in the figure) in antigenic
site Ca are coloured cyan. RBS indicates approximate location of
the receptor-binding site.
(TIFF)
Author Contributions
Conceived and designed the experiments: DMS GDS JAD MP. Performed
the experiments: SS AE KF JBG HC NB PVC MK MP YL. Analyzed the
data: NZJ DMS GDS SS AE KF JBG HC NB PVC MK MP YL. Wrote
the paper: DMS NZJ GDS SS AE JAD KF ALW JBG MK MP HC NB
TLK SMM PVC YL. Data collection: DMS NZJ GDS JAD ALW SMM
TLK. Literature search: TLK.
Table S1 Influenza A H3 and H1 antigenic site maps.
(PDF)
Table S2 Proportion of participants who received 2012–
13 TIV by age and influenza type, subtype and lineage.
(PDF)
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